Radiolabeled vasoactive intestinal peptides for diagnosis and therapy

ABSTRACT

This invention relates to radiotherapeutic reagents and peptides, radiodiagnostic reagents and peptides, and methods for producing labeled radiodiagnostic and radiotherapeutic agents. Specifically, the invention relates to vasoactive intestinal peptide receptor binding peptides, derivatives and analogues of vasoactive intestinal peptide, and embodiments of such peptides radiolabeled with a radioisotope, as well as methods and kits for making, radiolabeling and using such peptides for radiodiagnostic and radiotherapeutic purposes. The invention specifically relates to vasoactive intestinal peptide receptor binding peptide derivatives and analogues of vasoactive intestinal peptide radiolabeled with technetium-99m and uses thereof as scintigraphic imaging agents. The invention also specifically relates to vasoactive intestinal peptide receptor binding peptide derivatives and analogues of vasoactive intestinal peptide radiolabeled with cytotoxic radioisotopes such as rhenium-186 ( 186  Re) and rhenium-188 ( 188  Re) for use as radiotherapeutic agents. Methods and kits for making, radiolabeling and using such peptides diagnostically and therapeutically in a mammalian body are also provided.

This application is also a continuation-in-part of U.S. patentapplication Ser. No. 08/414,424, filed Mar. 31, 1995 and now U.S. Pat.No. 5,849,261.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to radiotherapeutic agents and peptides,radiodiagnostic agents and peptides, and methods for producing suchlabeled radiodiagnostic and radiotherapeutic agents. Specifically, theinvention relates to receptor-binding vasoactive intestinal peptides(including native vasoactive intestinal peptide (VIP) and fragments,derivatives, analogues and mimetics thereof), and embodiments of suchcompounds labeled with gamma-radiation emitting isotopes such astechnetium-99m (Tc-99m), as well as methods and kits for making,radiolabeling and using such peptides to image sites in a mammalianbody. The invention also relates to receptor binding vasoactiveintestinal peptides and derivatives, analogues and mimetics thereof,labeled with cytotoxic radioisotopes such as rhenium-186 (¹⁸⁸ Re) andrhenium-188 (¹⁸⁸ Re), and methods and kits for making, radiolabeling andusing such compounds therapeutically in a mammalian body.

2. Description of the Prior Art

Native vasoactive intestinal peptide (VIP) is a 28 amino acid peptidethat was first isolated from hog upper small intestine (Said and Mutt.1970, Science 169: 1217-1218). This peptide belongs to a family ofstructurally-related, small peptides that includes helodermin, secretin,the somatostatins, and glucagon. The peptide has the formula:

    HSDAVFTDNYTRLRKQMAVKKYLNSILN.amide (SEQ. ID NO.:1)         Formula I

(where single-letter abbreviations for amino acids can be found inZubay. Biochemistry 2d ed., 1988. MacMillan Publishing: New York, p.33).

The biological effects of VIP are mediated by the activation ofmembrane-bound receptor proteins that are coupled to the intracellularcyclic adenosine monophosphate signalling system. VIP regulates avariety of different biological activities in tissues and organs. Itmodulates cellular metabolic activities and regulates exocrine andendocrine secretions. It also induces relaxation of smooth muscle andcauses vasodilatory effects. VIP is also involved in the regulation ofcellular proliferation and survival in a number of different cell types,including keratinocytes, smooth muscle cells, sympathetic neuroblasts,hippocampal cells and, in vitro, NIH 3T3 cells.

VIP receptors are widely distributed throughout the gastrointestinaltract and are also found in various other cell types. Large numbers ofVIP receptors are expressed in rumor cells of, for example,adenocarcinomas, breast cancers, melanomas, neuroblastomas andpancreatic carcinomas. In fact, expression of high affinity bindingsites for VIP (comprising the VIP receptor protein) is a marker forthese tumor cells. Specific binding of VIP to these cells can beexploited as a marker to locate and identify such tumor cells in vivo.

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷ Ga, ^(99m) Tc (hereinafter Tc-99m), ¹¹¹ In, ¹²³ I, ¹²⁵ I,¹⁶⁹ Yb or ¹⁸⁶ Re. A number of factors must be considered for optimalradioimaging in humans. To maximize the efficiency of detection, aradionuclide that emits gamma energy in the 200 to 200 keV range ispreferred. To minimize the absorbed radiation dose to the patient, thephysical half-life of the radionuclide must be as short as the imagingprocedure will allow. To allow for examinations to be performed on anyday and at any time of the day, it is advantageous to have a source ofthe radionuclide always available at the clinical site.

Methods for radioiodinating VIP analogues at tyrosine residues in theVIP sequence (Tyr¹⁰ or Tyr²²) using ¹²³ I or ¹²⁵ I are known in theprior art. These radioiodinated species have also been used to assessVIP binding to receptors on tumor cells.

Boissard et al., 1986, Cancer Res. 46: 4406-4413 describeradioiodination of VIP and binding to human colon adenocarcinoma cells.

El Battari et al., 1988, J. Biol. Chem. 263: 17685-17689 describeraioiodination of VIP and binding to human colon adenocarcinoma cells.

Shaffer et al., 1987, Peptides 8: 1101-1106 disclose radioiodination ofVIP and binding to human small cell and non-small cell carcinoma cells.

Svoboda et al., 1988, Eur. J. Biochem. 176: 707-713 describeradioiodination of VIP and binding to rat transformed pancreatic acinarcells.

Gespach et al., 1988, Cancer Res. 48: 5079-5083 disclose radioiodinationof VIP and binding to human breast cancer cells.

Muller et al., 1989, J. Biol. Chem. 264: 3647-3650 discloseradioiodination of VIP and binding to human neuroblastoma cells.

Lee et al., 1990, Peptides 11: 1205-1210 disclose radioiodination of VIPand binding to human small cell and non-small cell carcinoma cells.

Park et al., 1990, Cancer Res. 50: 2773-2780 describe radioiodination ofVIP and binding to human gastric cancer cells.

Bellan et al., 1992, Exp. Cell Res. 200: 34-40 disclose radioiodinationof VIP and binding to human melanoma cells.

Moody et al., 1993, Proc. Natl. Acad. Sci. USA 90: 4345-4349 discloseradioiodination of VIP and binding to non-small cell lung carcinomacells.

Virgolini et al., 1994, Cancer Res. 54: 690-700 describe radioiodinationof VIP and binding to primary tumors and tumor cell lines.

Methods for radioiodinating VIP analogues at Y¹⁰ or Y²² using ¹³¹ I areknown in the prior art.

Hassan et al., 1994, Nucl. Med. Biol. 21: 865-872 discloseradioiodination of VIP and in vivo distribution of radiolabel in a ratby scintigraphy.

These methods have application for enabling detection of tumor cells invivo by radioimaging, particularly radioscintigraphy. VIP is a usefulmarker for such radioimaging, because many different tumor cells expressa high affinity binding site for VIP (the VIP receptor protein).However, radioiodinated peptides have significant commercialdisadvantages. ¹²³ I is both expensive and in limited supply. Also,approved radioiodinated radiopharmaceuticals normally cannot be preparedat the clinical site.

Tc-99m is a preferred radionuclide because it emits gamma radiation at140 keV, it has a physical half-life of 6 hours, and it is readilyavailable on-site using a molybdenum-99/technetium-99m generator. Otherradionuclides used in the prior art are less advantageous than Tc-99m.This can be because the physical half-lives of such radionuclides arelonger, resulting in a greater amount of absorbed radiation dose to thepatient (e.g., indium-111). Alternatively, the gamma radiation energiesof such alternate radionuclides are significantly lower (e.g.,iodine-125) or higher (e.g., iodine-131) than Tc-99m and are therebyinappropriate for quality scintigraphic imaging. Furthermore, manydisadvantageous radionuclides cannot be produced using an on-sitegenerator.

Tc-99m is a transition metal that is advantageously chelated by a metalcomplexing moiety. Radiolabel complexing moieties capable of bindingTc-99m can be covalently linked to various specific binding compounds toprovide a means for radiolabeling such specific binding compounds. Thisis because the most commonly available chemical species of Tc-99m,pertechnetate (TcO₄ ⁻), cannot bind directly to most specific bindingcompounds strongly enough to be useful as a radiopharmaceutical.Complexing of Tc-99m with such radiolabel complexing moieties typicallyentails chemical reduction of the pertechnetate using a reducing agentsuch as stannous chloride.

Although Tc-99m is the preferred radionuclide for scintigraphic imaging,it has not been widely used for labeling peptides (see Lamberts, 1991,J. Nucl. Med. 32: 1189-1191). This is because methods known in the priorart for labeling larger protein molecules (i.e., >10,000 daltons insize) with Tc-99m are not suitable for labeling peptides (having amolecular size less than 10,000 daltons). Consequently, it is necessaryto radiolabel most peptides by covalently attaching a radionuclidechelating moiety to the peptide, so that the chelator is incorporatedsite-selectively at a position in the peptide that will not interferewith the specific binding properties of the peptide.

Methods for labeling peptides with Tc-99m are disclosed in co-owned U.S.Pat. No. 5,225,180 and in co-pending U.S. patent application Ser. No.07/653,012, now abandoned, which issued as U.S. Pat. No. 5,811,394; Ser.No. 07/807,062, now U.S. Pat. No. 5,443,815; Ser. No. 07/851,074, nowabandoned, a divisional of which issued as U.S. Pat. No. 5,711,931; Ser.No. 07/871,282, a divisional of which issued as U.S. Pat. No. 5,720,934;Ser. No. 07/886,752, now abandoned, which issued as U.S. Pat. No.5,849,260; Ser. No. 07/893,981, now U.S. Pat. No. 5,508,020; Ser. No.07/902,935, now U.S. Pat. No. 5,716,596; Ser. No. 07/955,466, nowabandoned; Ser. No. 07/977,628, now U.S. Pat. No. 5,405,597; Ser. No.08/019,864, now U.S. Pat. No. 5,552,525; Ser. No. 08/044,825, nowabandoned, which issued as U.S. Pat. No. 5,645,815; Ser. No. 08/073,577,now U.S. Pat. No. 5,561,220; Ser. Nos. 08/092,355; 08/095,760, now U.S.Pat. No. 5,620,675; and Ser. No. 08/210,822, now abandoned, and PCTInternational Applications PCT/US92/00757, PCT/US92/10716,PCT/US93/02320, PCT/US93/03687, PCT/US93/04794, PCT/US93/05372,PCT/US93/06029, PCT/US93/09387, and PCT/US94/01894, which are herebyincorporated by reference. However none of these references disclosespecifically how to prepare technetium-99m -labeled vasoactiveintestinal peptides.

Methods for preparing Tc-99m complexes are known in the art and examplesare provided below for general reference:

Byrne et al., U.S. Pat. Nos. 4,434,151, 4,575,556 and 4,571,430 describehomocysteine thiolactone-derived bifunctional chelating agents.

Fritzberg, U.S. Pat. No. 4,444,690 describes a series oftechnetium-chelating agents based on2,3-bis(mercaptoacetamido)propanoate.

Nosco et al., U.S. Pat. No. 4,925,650 describe Tc-99m chelatingcomplexes.

Kondo et al., European Patent Application, Publication No. 483704 A1disclose a process for preparing a Tc-99m complex with amercapto-Gly-Gly-Gly moiety.

European Patent Application No. 84109831.2 describes bisamido, bisthiolTc-99m ligands and salts thereof as renal function monitoring agents.

Davison et al., 1981, Inorg. Chem. 20: 1629-1632 disclose oxotechnetiumchelate complexes.

Fritzberg et al., 1982, J. Nucl. Med. 23: 592-598 disclose a Tc-99mchelating agent based on N,N'-bis(mercaptoacetyl)-2,3-diaminopropanoate.

Byrne et al., 1983, J. Nucl. Med. 24: P126 describehomocystine-containing Tc-99m chelating agents.

Bryson et al., 1988, Inorg. Chem. 27: 2154-2161 describe neutralcomplexes of technetium-99 which are unstable to excess ligand.

Misra et al., 1989, Tet. Lett. 30: 1885-1888 describe bisamine bisthiolcompounds for radiolabeling purposes.

The use of chelating agents for radiolabeling specific-binding compoundsis known in the art and examples are provided below for generalreference:

Gansow et al., U.S. Pat. No. 4,472,509 teach methods of manufacturingand purifying Tc-99m chelate-conjugated monoclonal antibodies.

Stavrianopoulos, U.S. Pat. No. 4,943,523 teach detectable moleculescomprising metal chelating moieties.

Fritzberg et al., European Patent Application No. 86100360.6 describedithiol, diamino, or diamidocarboxylic acid or amine complexes usefulfor making technetium-labeled imaging agents.

Albert et al., UK Patent Application 8927255.3 disclose radioimagingusing somatostatin derivatives such as octreotide labeled with ¹¹¹ I viaa chelating group bound to the amino-terminus.

Albert et al., European Patent Application No. WO 91/01144 discloseradioimaging using radiolabeled peptides related to growth factors,hormones, interferons and cytokines and comprised of a specificrecognition peptide covalently linked via an amino group of said peptideto a radionuclide chelating group.

Fischman et al., International Patent Application, Publication No.WO93/13317 disclose chemotactic peptides attached to chelating moieties.

Kwekkeboom et al., 1991, J. Nucl. Med. 32: 981 Abstract #305 relates toradiolabeling somatostatin analogues with ¹¹¹ In.

Albert et al., 1991, Abstract LM10, 12th American Peptide Symposium:1991 describe uses for ¹¹¹ In-labeled diethylene-triaminopentaaceticacid-derivatized somatostatin analogues.

Cox et al., 1991, Abstract. 7th International Symposium onRadiopharmacology, p. 16, disclose the use of, Tc-99m-, ¹³¹ I- and ¹¹¹In-labeled somatostatin analogues in radiolocalization of endocrinetumors in vivo by scintigraphy.

Methods for labeling certain specific-binding compounds, mainly largeproteins, with Tc-99m are known in the prior art, and examples areprovided below for general reference:

Hnatowich, U.S. Pat. No. 4,668,503 describe Tc-99m proteinradiolabeling.

Tolman, U.S. Pat. No. 4,732,684 describe conjugation of targetingmolecules and fragments of metallothionein.

Nicolotti et al., U.S. Pat. No. 4,861,869 describe bifunctional couplingagents useful in forming conjugates with biological molecules such asantibodies.

Fritzberg et al., U.S. Pat. No. 4,965,392 describe various S-protectedmercaptoacetylglycylglycine-based chelators for labeling proteins.

Schochat et al., U.S. Pat. No. 5,061,641 disclose direct radiolabelingof proteins comprised of at least one "pendent" sulfhydryl group.

Fritzberg et al., U.S. Pat. No. 5,091,514 describe various S-protectedmercaptoacetylglycylglycine-based chelators for labeling proteins.

Gustavson et al., U.S. Pat. No. 5,112,953 disclose Tc-99m chelatingagents for radiolabeling proteins.

Kasina et al., U.S. Pat. No. 5,175,257 describe various combinations oftargeting molecules and Tc-99m chelating groups.

Dean et al., U.S. Pat. No. 5,180,816 disclose methods for radiolabelinga protein with Tc-99m via a bifunctional chelating agent.

Sundrehagen, International Patent Application, Publication No.WO85/03231 disclose Tc-99m labeling of proteins.

Reno and Bottino, European Patent Application 87300426.1 discloseradiolabeling antibodies with Tc-99m.

Bremer et al., European Patent Application No. 87118142.6 discloseTc-99m radiolabeling of antibody molecules.

Pak et al., European Patent Application No. WO 88/07382 disclose amethod for labeling antibodies with Tc-99m.

Goedemans et al., PCT Application No. WO 89/07456 describe radiolabelingproteins using cyclic thiol compounds, particularly 2-iminothiolane andderivatives.

Dean et al., International Patent Application, Publication No.WO89/12625 teach bifunctional coupling agents for Tc-99m labeling ofproteins.

Schoemaker et al., International Patent Application, Publication No.WO90/06323 disclose chimeric proteins comprising a metal-binding region.

Thornback et al., EPC Application No. 90402206.8 describe preparationand use of radiolabeled proteins or peptides using thiol-containingcompounds, particularly 2-iminothiolane.

Gustavson et al., International Patent Application, Publication No.WO91/09876 disclose Tc-99m chelating agents for radiolabeling proteins.

Rhodes, 1974, Sem. Nucl. Med. 4: 281-293 teach the labeling of humanserum albumin with technetium-99m.

Khaw et al., 1982, J. Nucl. Med. 23: 1011-1019 disclose methods forlabeling biologically active macromolecules with Tc-99m.

Schwartz et al., 1991, Bioconjugate Chem. 2: 333 describe a method forlabeling proteins with Tc-99m using a hydrazinonicotinamide group.

Attempts at labeling peptides have been reported in the prior art.

Ege et al., U.S. Pat. No. 4,832,940 teach radiolabeled peptides forimaging localized T-lymphocytes.

Morgan et al., U.S. Pat. No. 4,986,979 disclose methods for imagingsites of inflammation.

Flanagan et al., U.S. Pat. No. 5,248,764 describe conjugates between aradiolabel chelating moiety and atrial natiuretic factor-derivedpeptides.

Ranby et al., 1988, PCT/US88/02276 disclose a method for detectingfibrin deposits in an animal comprising covalently binding aradiolabeled compound to fibrin.

Lees et al., 1989, PCT/US89/01854 teach radiolabeled peptides forarterial imaging.

Morgan et al., International Patent Application, Publication No.WO90/10463 disclose methods for imaging sites of inflammation.

Flanagan et al., European Patent Application No. 90306428.5 discloseTc-99m labeling of synthetic peptide fragments via a set of organicchelating molecules.

Stuttle, PCT Application, Publication No. WO 90/15818 suggests Tc-99mlabeling of RGD-containing oligopeptides.

Rodwell et al., 1991, PCT/US91/03116 disclose conjugates of "molecularrecognition units" with "effector domains".

Cox, International Patent Application No. PCT/US92/04559 disclosesradiolabeled somatostatin derivatives containing two cysteine residues.

Rhodes et al., International Patent Application, Publication No.WO93/12819 teach peptides comprising metal ion-binding domains.

Lyle et al, International Patent Application, Publication No. WO93/15770disclose Tc-99m chelators and peptides labeled with Tc-99m.

Coughlin et al, International Patent Application, Publication No.WO93/21151 disclose bifunctional chelating agents comprising thioureagroups for radiolabeling targeting molecules.

Knight et al., 1990, 37th Annual Meeting of the Society of NuclearMedicine, Abstract #209, claim thrombus imaging using Tc-99m labeledpeptides.

Babich et al., 1993, J. Nucl. Med. 34: 1964-1974 describe Tc-99m labeledpeptides comprising hydrazinonicotinamide derivatives.

Radiolabeled derivatives of VIP receptor binding peptides and fragments,analogues and mimetics thereof can also be used therapeutically. Forthese applications, cytotoxic radioisotopes rhenium-186 and rhenium-188are particularly advantageous.

Thus there remains a need for synthetic (to make routine manufacturepracticable and to ease regulatory acceptance) VIP receptor bindingpeptides. derivatives, analogues and mimetics which can be radiolabeledwith Tc-99m, for use as scintigraphic agents and which can beradiolabeled with rhenium-186 or henium-188 for use as radiotherapeuticagents. Small synthetic VIP receptor binding peptides, derivatives,analogues and mimetics thereof which contain chelating groups forchelating Tc-99m, Re-186 or Re-188 and their Tc-99m, Re-186 and Re-188labeled derivatives are provided by this invention that specificallyfulfill this need.

SUMMARY OF THE INVENTION

The present invention provides radiopharmaceuticals that are Tc-99m,Re-186 or Re-188 labeled receptor binding vasoactive intestinalvasoactive intestinal peptides for radiotherapeutic applications andradiodiagnostic applications, in particular scintigraphic imagingapplications. The invention also provides receptor binding vasoactiveintestinal peptide reagents comprised of the receptor binding vasoactiveintestinal peptides, derivatives, analogues and mimetics thereof,wherein such compounds are covalently linked to a chelating moiety. Theinvention provides such receptor binding vasoactive intestinal peptides,receptor binding vasoactive intestinal peptide reagents and radiolabeledreceptor binding vasoactive intestinal peptide reagents that arescintigraphic imaging agents, radiodiagnostic agents andradiotherapeutic agents.

Scintigraphic imaging agents of the invention comprise receptor bindingvasoactive intestinal peptide reagents radiolabeled with technetium-99m.Radiotherapeutic agents of the invention comprise receptor bindingvasoactive intestinal peptide reagents radiolabeled with rhenium-186 orrhenium-188. Methods for making and using such receptor bindingvasoactive intestinal peptides, receptor binding vasoactive intestinalpeptide reagents and radiolabeled embodiments thereof are also provided.

The invention provides a reagent for preparing a radiopharmaceutical,wherein the reagent is a synthetic, receptor-binding vasoactiveintestinal peptide (defined as a synthetic compound including VIP,fragments, derivatives, analogues and mimetics thereof, which binds to aVIP receptor) that is covalently linked to a chelating moiety capable ofchelating a technetium or rhenium radiolabel. The chelating moiety isincorporated into the reagent during synthesis of the reagent. Inaddition, the technetium- or rhenium-labeled radiopharmaceuticals of theinvention have a VIP receptor binding affinity that is not less thanabout one-tenth the affinity of radioiodinated native VIP. In apreferred embodiment, the invention provides scintigraphic imagingagents comprising a reagent of the invention radiolabeled with Tc-99m.In other preferred embodiments, the invention provides radiotherapeuticagents comprising a reagent of the invention radiolabeled with acytotoxic radioisotope selected from the group consisting of rhenium-186and rhenium-188. Complexes of the reagent and radiolabels that areTc-99m, Re-186 or Re-188 are formed by reacting a reagent of theinvention with the radiolabel in the presence of a reducing agent, forexample, a stannous ion. Complexes of Tc-99m, Re-186 or Re-188 with thereagents of the invention are also provided as produced by ligandexchange of a prereduced radiolabel complex.

Thus, the invention also provides scintigraphic imaging agentscomprising the receptor binding vasoactive intestinal peptide reagentsof the invention wherein the chelating moiety is stably complexed withTc-99m.

The invention also provides radiotherapeutic agents that are thereceptor binding vasoactive intestinal peptide reagents of the inventionradiolabeled with rhenium-186 or rhenium-188.

The invention also provides pharmaceutical compositions comprising theradiolabeled receptor-binding vasoactive intestinal peptides of theinvention in a pharmaceutically acceptable carrier.

Another aspect of the present invention provides reagents for preparingradiotherapeutic and radiodiagnostic radiopharmaceuticals, includingpreferably scintigraphic imaging agents. Each such reagent is comprisedof a compound that is vasoactive intestinal peptide, derivative, analogor mimetic covalently linked to a chelating moiety.

A first aspect of the reagents provided by the invention for preparingradiolabeled agents are reagents that are each comprised of areceptor-binding vasoactive intestinal peptide as described above thatis covalently linked to a chelating moiety having the formula:

    C(pgp).sup.S --(aa)--C(pgp).sup.S

where (pgp)^(S) is hydrogen or a thiol protecting group and (aa) is anα- or β-amino acid not comprising a thiol group. In a preferredembodiment, the amino acid is glycine. In another preferred embodiment,the agent is a scintigraphic imaging agent. In another preferredembodiment, the agent is a radiotherapeutic agent.

In a second embodiment, the invention provides receptor bindingvasoactive intestinal peptide reagents capable of being radiolabeled toform radiodiagnostic and radiotherapeutic agents, each comprising avasoactive intestinal peptide covalently linked to a chelating groupcomprising a single thiol containing moiety having the formula:

    A--CZ(B)--{C(R.sup.a R.sup.b))}.sub.n --X

wherein A is H, HOOC, H₂ NOC, (peptide)-NHOC, (peptide)-OOC, R^(a) NCO,or R^(d) ; B is H, SH or --NHR^(c), --N(R^(c))-(peptide) or R^(d) ; Z isH or R^(d) ; X is SH or --NHR^(c), --N(R^(c))-(peptide) or R^(d) ;R^(a), R^(b), R^(c) and R^(d) are independently H or straight orbranched chain or cyclic lower alkyl; n is 0, 1 or 2; R^(c) is C₁ -C₄alkyl, an amino acid or a peptide comprising 2 to about 10 amino acids,and: (1) where B is --NHR or --N(R^(c))-(peptide), X is SH and n is 1 or2; (2) where X is --NHR or --N(R^(c))-(peptide), B is SH and n is 1 or2; (3) where B is H or R^(d), A is HOOC, H₂ NOC, (peptide)-NHOC, or(peptide)-OOC, X is SH and n is 0 or 1; (4) where A is H or R^(c), thenwhere B is SH, X is --NHR^(c) or --N(R^(c))-(peptide) and where X is SH,B is --NHR^(c) or --N(R^(c))-(peptide) and n is 1 or 2; (5) where X is Hor R^(d), A is HOOC, H₂ NOC, (peptide)-NHOC, or (peptide)-OOC and B isSH; (6) where Z is methyl, X is methyl, A is HOOC, H₂ NOC,(peptide)-NHOC, or (peptide)-OOC and B is SH and n is 0. In a preferredembodiment, the agent is a scintigraphic imaging agent. In yet anotherpreferred embodiment, the agent is a radiotherapeutic agent.

Preferred embodiments of this chelating moiety have a chemical formulathat is:

    R.sup.1 --CO-(amino acid).sup.1 -(amino acid).sup.2 -Z

wherein (amino acid)¹ and (amino acid)² are each independently anyprimary a- or b-amino acid that does not comprise a thiol group, Z is athiol-containing moiety that is cysteine, homocysteine, isocysteine,penicillamine, 2-mercaptoethylamine or 3-mercaptopropylamine, and R¹ islower (C¹ -C⁴)alkyl, an amino acid or a peptide comprising 2 to 10 aminoacids. When Z is cysteine, homocysteine, isocysteine or penicillamine,the carbonyl group of said moiety is covalently linked to a hydroxylgroup, a NR³ R⁴ group, wherein each of R³ and R⁴ are independently H orlower (C¹ -C⁴) alkyl, an amino acid or a peptide comprising 2 to 10amino acids; or

    Y-(amino acid).sup.2 -(amino acid).sup.1 -NHR.sup.2

wherein Y is a thiol-containing moiety that is cysteine, homocysteine,isocysteine, penicillamine, 2-mercaptoacetate or 3-mercaptopropionate,(amino acid)¹ and (amino acid)² are each independently any primary a- orb-amino acid that does not comprise a thiol group, and R² is H or lower(C¹ -C⁴)alkyl, an amino acid or a peptide comprising 2 to 10 aminoacids. When Y is cysteine, homocysteine, isocysteine or penicillamine,the amino group of said moiety is covalently linked to --H, an aminoacid or a peptide comprising 2 to 10 amino acids.

In particular embodiments of this aspect of the invention, the chelatingmoiety has a formula that is:

IIa. -(amino acid)¹ -(amino acid)² -A--CZ(B)--{C(R¹ R²)}_(n) --X),

IIb. --A--CZ(B)--{C(R¹ R²)}_(n) --X}-(amino acid)¹ -(amino acid)²,

IIc. -(a primary α,β- or β-γ-diamino acid)-(amino acid)¹-A--CZ(B)--{ε(R¹ R²)})_(n) --X}, or

IId. --A--CZ(B)--{C(R¹ R²)}_(n) --X}-(amino acid)¹ -(a primary a.β orβ,γ-diamino acid) wherein (amino acid)¹ and (amino acid)² are eachindependently any naturally-occurring, modified, substituted or altereda- or b-amino acid not containing a thiol group: A is H, HOOC, H₂ NOC,(amino acid or peptide)-NHOC, (amino acid or peptide)-OOC or R⁴ ; B isH, SH or --NHR³, --N(R³)-(amino acid or peptide) or R⁴ ; Z is H or R⁴ ;X is SH or --NHR³, --N(R³)-(amino acid or peptide) or R⁴ ; R¹, R², R³and R⁴ are independently H or straight or branched chain or cyclic loweralkyl; n is an integer that is either 0, 1 or 2; (peptide) is a peptideof 2 to about 10 amino acids: and: (1) where B is --NHR³ or--N(R³)-(amino acid or peptide), X is SH and n is 1 or 2; (2) where X is--NHR³ or --N(R³)-(amino acid or peptide), B is SH and n is 1 or 2; (3)where B is H or R⁴, A is HOOC, H₂ NOC, (amino acid or peptide)-NHOC,(amino acid or peptide)-OOC, X is SH and n is 0 or 1; (4) where A is Hor R⁴, then where B is SH, X is --NHR³ or --N(R³)-(amino acid orpeptide) and where X is SH, B is --NHR³ or --N(R³)-(amino acid orpeptide) and n is 1 or 2; (5) where X is H or R⁴, A is HOOC, H₂ NOC,(amino acid or peptide)-NHOC, (amino acid or peptide)-OOC and B is SH;(6) where Z is methyl, X is methyl, A is HOOC, H₂ NOC. (amino acid orpeptide)-NHOC, (amino acid or peptide)-OOC and B is SH and n is 0.

Additional preferred embodiments include chelating moieties having theformula: -Gly-Gly-Cys-, Cys-Gly-Gly-, Gly-Gly-Cys-, -(ε-Lys)-Gly-Cys-.(δ-Orn)-Gly-Cys-, -(γ-Dab)-Gly-Cys-, -(β-Dap)-Lys-Cys- a-(β-Dap)-Gly-Cys-. (In these formulae, it will be understood that ε-Lysrepresents a lysine residue in which the ε-amino group, rather than thetypical α-amino group, is covalently linked to the carboxyl group of theadjacent amino acid to form a peptide bond: δ-Orn represents anornithine residue in which the δ-amino group, rather than the typicalα-amino group, is covalently linked to the carboxyl group of theadjacent amino acid to form a peptide bond: γ-Dab represents a2,4-diaminobutyric acid residue in which the γ-amino group is covalentlylinked to the carboxyl group of the adjacent amino acid to form apeptide bond; and β-Dap represents a 1,3-diaminopropionic acid residuein which the β-amino group is covalently linked to the carboxyl group ofthe adjacent amino acid to form a peptide bond.)

Yet another embodiment of the invention provides receptor bindingvasoactive intestinal peptide reagents capable of being radiolabeledwith a radioisotope for imaging sites within a mammalian body or forradiotherapeutic purposes, each comprising a vasoactive intestinalpeptide that is covalently linked to a chelating moiety that is abisamino-bisthiol chelating moiety. The bisamino bisthiol chelatingmoiety in this embodiment of the invention has the formula: ##STR1##wherein each R can be independently H, CH₃ or C₂ H₅ ; each (pgp)^(S) canbe independently a thiol protecting group or H; m, n and p areindependently 2 or 3; A is linear or cyclic lower alkyl aryl,heterocyclyl, combinations or substituted derivatives thereof; and X ispeptide; or ##STR2## wherein each R is independently H, CH₃ or C₂ H₅ ;m, n and p are independently 2 or 3; A is linear or cyclic lower alkyl,aryl, heterocyclyl, combinations or substituted derivatives thereof; Vis H or CO-peptide: R' is H or peptide, provided that when V is H, R' ispeptide and when R' is H, V is peptide. For purposes of this invention,chelating moieties having these structures will be referred to as "BAT"moieties. In a preferred embodiment, the agent is a scintigraphicimaging agent. In yet another preferred embodiment, the agent is aradiotherapeutic agent.

The invention also provides radiopharmaceutical agents and reagents forpreparing such radiopharmaceuticals comprising a receptor bindingvasoactive intestinal peptide covalently linked to a chelating moietyselected from the group consisting of:

(i) a group having the formula: ##STR3## (ii) a group having theformula: ##STR4## wherein n, m and p are each integers that areindependently 0 or 1; each R' is independently H, lower alkyl, C₂ -C₄hydroxyalkyl, or C₂ -C₄ alkoxyalkyl, and each R is independently H orR", where R" is substituted or unsubstituted lower alkyl or phenyl notcomprising a thiol group, and one R or R' is L, where L is a bivalentlinker moiety linking the metal chelator to the targeting moiety andwherein when one R' is L, NR'₂ is an amine.

In preferred embodiments, L is a C₁ -C₆ linear, branched chain or cyclicalkyl group, a carboxylic ester, a carboxamide, a sulfonamide, an ether,a thioether, an amine, an alkene, an alkyne, a 1,2-. 1,3- or 1,4-linked,optionally substituted, benzene ring, or an amino acid or peptide of 2to about 10 amino acids, or combinations thereof.

In preferred embodiments, R" is a C₁ -C₆ linear, branched or cyclicalkyl group; a --C_(q) OC_(r) --, --C_(q) NHC_(r) -- or --C_(q) SC_(r)-- group, where q and r are integers each independently 1 to 5 whereinthe sum of q+r is not greater than 6; (C₁ -C₆) alkyl-X. where X is ahydroxyl group, a substituted amine, a guanidine, an amidine, asubstituted thiol group, or a carboxylic acid, ester, phosphate, orsulfate group; a phenyl group or a phenyl group substituted with ahalogen, hydroxyl, substituted amine, guanidine, amidine, substitutedthiol, ether, phosphate, or sulfate group; an indole group; a C₁ -C₆heterocyclic group containing 1 to 3 nitrogen, oxygen or sulfur atoms orcombinations thereof.

Preferred chelating moieties of the invention include chelators havingthe formula: ##STR5## wherein R¹ and R² are each independently H, loweralkyl, C₂ -C₄ hydroxyalkyl, or C₂ -C₄ alkoxyalkyl; R³, R⁴, R⁵ and R⁶ areindependently H, substituted or unsubstituted lower alkyl or phenyl notcomprising a thiol group: R⁷ and R⁸ are each independently H, loweralkyl, lower hydroxyalkyl or lower alkoxyalkyl; L is a bivalent linkergroup and Z is a vasoactive intestinal peptide.

Additional preferred metal chelators of the invention include chelatorsof formula: ##STR6## wherein R¹ and R² are each independently H, loweralkyl, C₂ -C₄ hydroxyalkyl, or C₂ -C₄ alkoxyalkyl; R³, R⁴, R⁵ and R⁶ areindependently H, substituted or unsubstituted lower alkyl or phenyl notcomprising a thiol group, and one of R³, R⁴. R⁵ or R⁶ isZ--L--HN(CH₂)_(n) --, where L is a bivalent linker group, Z is atargeting moiety, and n is an integer from 1 to 6; R⁷ and R⁸ are eachindependently H, lower alkyl, lower hydroxyalkyl or lower alkoxyalkyl;and X is an amino group, a substituted amino group or --NR¹ --Y, where Yis an amino acid, an amino acid amide, or a peptide comprising from 2 to10 amino acids.

More preferred metal chelators of the invention include chelators havingthe formula: ##STR7## wherein R¹ and R² are each independently H, loweralkyl, lower hydroxyalkyl, or lower alkenylalkyl; R³ and R⁴ areindependently H, substituted or unsubstituted lower alkyl or phenyl notcomprising a thiol group; n is an integer from 1 to 6; L is a bivalentlinker group; and Z is a vasoactive intestinal peptide moiety.

Additional more preferred chelating moieties include chelators offormula: ##STR8## wherein L is a bivalent linker group and Z is avasoactive intestinal peptide moiety.

Most preferred chelating moieties of the invention include chelatorshaving the following formulae:

(amino acid)¹ -(amino acid)² -cysteine-,

(amino acid)¹ -(amino acid)² -isocysteine-.

(amino acid)¹ -(amino acid)² -homocysteine-,

(amino acid)¹ -(amino acid)² -penicillamine-,

(amino acid)¹ -(amino acid)² -2-mercaptoethylamine-,

(amino acid)¹ -(amino acid)² -2-mercaptopropylamine-,

(amino acid)¹ -(amino acid)² -2-mercapto-2-methylpropylamine-,

(amino acid)¹ -(amino acid)² -3-mercaptopropylamine-,

wherein (amino acid) in a primary α- or β-amino acid not comprising athiol group and wherein the chelator is attached to either a targetingmoiety or a linker group via a covalent bond with the carboxyl terminusof the chelator or a side chain on one of the amino acid groups.

Most preferred chelators also include chelators of the above formulawherein (amino acid)¹ is either an α,γ- or β,γ-amino acid wherein the α-or β-amino group is a free amine and the α,γ- or β-γ-amino acid iscovalently linked via the γ amino group.

Other most preferred chelators include those selected from the groupconsisting of:

-cysteine-(amino acid)-(α,β- or β,γ-diamino acid);

-isocysteine-(amino acid)-(α,β- or β,γ-diamino acid);

-homocysteine-(amino acid)-(α,β- or β,γ-diamino acid);

-penicillamine-(amino acid)-(α,β- or β,γ-diamino acid);

2-mercaptoacetic acid-(amino acid)-(α,β- or β,γ-diamino acid);

2- or 3-mercaptopropionic acid-(amino acid)-(α,β- or β,γ-diamino acid);

2-mercapto-2-methylpropionic acid-(amino acid)-(α,β- or β,γ-diaminoacid);

wherein (amino acid) in a primary α- or β-amino acid not comprising athiol group and wherein the chelator is attached to either a targetingmoiety or a linker group via a covalent bond with the amino terminus ofthe chelator or a side chain on one of the amino acid groups.

Particularly preferred metal chelators are selected from the groupconsisting of: Gly-Gly-Cys-, Arg-Gly-Cys-, -(ε-Lys)-Gly-Cys-,-(δ-Orn)-Gly-Cys-, -(γ-Dab)-Gly-Cys-, -(β-Dap)-Lys-Cys- and-(β-Dap)-Gly-Cys-. (In these formulae, it will be understood that: ε-Lysrepresents a lysine residue in which the ε-amino group, rather than thetypical α-amino group, is covalently linked to the carboxyl group of theadjacent amino acid to form a peptide bond; δ-Orn represents anornithine residue in which the δ-amino group, rather than the typicalα-amino group, is covalently linked to the carboxyl group of theadjacent amino acid to form a peptide bond; γ-Dab represents a2,4diaminobutyric acid residue in which the γ-amino group is covalentlylinked to the carboxyl group of the adjacent amino acid to form apeptide bond; and β-Dap represents a 1,3-diaminopropionic acid residuein which the β-amino group is covalently linked to the carboxyl group ofthe adjacent amino acid to form a peptide bond.)

An example of preferred chelating moieties of structure type (III) aboveis the chelator Gly-Gly-Cys- which forms a chelating moiety having thestructure: ##STR9##

Chelating ligands having structure type VII form oxotechnetium complexeshaving the structure: ##STR10##

An example of more preferred chelating moieties having structure type Vas shown above is Lys-(ω-peptide)-Gly-Cys.amide which forms a chelatingmoiety of structure: ##STR11##

Chelating ligands having structure type IX form oxotechnetium complexeshaving the structure: ##STR12##

An example of a reagent for preparing a radiopharmaceutical agent asprovided by this invention comprising a chelating moiety havingstructure type II as shown above is (targetingmoiety)-Cys-Gly-α,β-diaminopropionamide which forms a chelating moietyof structure: ##STR13##

Radiodiagnostic agents having structure type XI form oxotechnetiumcomplexes having the structure: ##STR14##

In preferred embodiments of the invention, the chelating moieties arecovalently attached to the receptor binding vasoactive intestinalpeptides other than at amino groups comprising the peptide. As describedherein, there are particular advantages to incorporating chelatingmoieties into the peptide during synthesis or to incorporating a uniqueattachment moiety for the chelating moiety into the peptide duringchemical synthesis. Amino groups in the side-chains of lysine residuesare present in vasoactive intestinal peptides st several positions andtherefore do not allow the advantages of a site-specific attachment of achelating moiety. In particular, thiol groups are preferred as uniqueattachment sites for attachment of chelators to the receptor bindingvasoactive instestinal peptides.

This invention also provides methods for preparing peptide reagents ofthe invention by chemical synthesis in vitro. In a preferred embodiment,receptor binding vasoactive intestinal peptides are synthesized by solidphase peptide synthesis.

This invention provides reagents for preparing a radiolabeled vasoactiveintestinal receptor-binding agent comprising the receptor bindingvasoactive intestinal peptides of the invention covalently linked to achelating moiety. In a preferred embodiment, the reagent isradioactively labeled with Tc-99m. In another preferred embodiment, thereagent is radioactively labeled with ¹⁸⁶ Re or ¹⁸⁸ Re.

The invention also comprises agents that are complexes of thereceptor-binding vasoactive intestinal peptide reagents of the inventionwith a radioisotope, as well as methods for radiolabeling the peptidereagents of the invention. For example, scintigraphic imaging agentsprovided by the invention comprise Tc-99m labeled complexes formed byreacting the vasoactive intestinal peptide reagents of the inventionwith Tc-99m in the presence of a reducing agent. Preferred reducingagents include but are not limited to dithionite ion, stannous ion andferrous ion. Such Tc-99m complexes of the invention are also formed bylabeling the vasoactive intestinal peptide reagents of the inventionwith Tc-99m by ligand exchange of a prereduced Tc-99m complex asprovided herein.

The invention also provides kits for preparing radiolabeled receptorbinding vasoactive intestinal peptides from the vasoactive intestinalpeptide reagents of the invention. Kits for radiolabeling the peptidereagents of the invention are comprised of a sealed vial containing apredetermined quantity of a peptide reagent of the invention and asufficient amount of reducing agent to radiolabel the reagent. In oneaspect of preferred embodiments of the kits of the invention are kitsfor radiolabeling the peptide reagents of the invention with Tc-99m.Kits for preparing radiotherapeutic agents are also provided, whereinthe preferred radioisotopes are rhenium-186 and rhenium-188.

This invention provides methods for using the radiolabeled vasoactiveintestinal receptor-binding peptide reagents of the inventiondiagnostically and therapeutically. In one embodiment of the invention,methods are provided for using scintigraphic imaging agents that areTc-99m labeled peptide reagents for imaging sites within a mammalianbody by obtaining in vivo gamma scintigraphic images. These methodscomprise administering an effective diagnostic amount of radiolabeledpeptide reagents of the invention and detecting the gamma radiationemitted by the radiolabel localized at the site within the mammalianbody.

The invention also provides methods for alleviating VIP-related diseasesin animals, preferably humans, comprising administering atherapeutically effective amount of the radiolabeled VIPreceptor-binding peptide reagents of the invention to the animal. Inpreferred embodiments, the reagent is radioactively labeled with ¹⁸⁶ Reor ¹⁸⁸ Re.

This invention also provides VIP receptor-binding peptides covalentlylinked to a metal-binding moiety that are complexed with a magnetic,paramagnetic, supermagnetic, or superparamagnetic metal atom, ion orparticle, and methods for using such complexes for magnetic-baseddetection of localization of such receptor binding vasoactive intestinalpeptide complexes at tumor or other tissue sites in vivo. Thus, theinvention provides non-radioactive methods for localizing rumor andother VIP receptor expressing tissues in vivo.

The receptor binding vasoactive intestinal peptides and receptor bindingvasoactive intestinal peptide reagents of the invention may also becomprised of a polyvalent linking moiety. Polyvalent linking moieties ofthe invention are comprised of at least 2 identical linker functionalgroups capable of covalently bonding to receptor binding vasoactiveintestinal peptides or chelating moieties or both. Preferred linkerfunctional groups are primary or secondary amines, hydroxyl groups,carboxylic acid groups or thiol-reactive groups. In preferredembodiments, the polyvalent linking moieties are comprised ofbis-succinimidylmethylether (BSME). 4-(2,2-dimethylacetyl)benzoic acid(DMBA), N-{2-(N',N'-bis(2-succinimido-ethyl)aminoethyl)}-N⁶,N⁹-bis(2-methyl-2-mercapto-propyl)-6,9-diazanonanamide (BAT-BS),tris(succinimidylethyl)amine (TSEA). bis-succinimidohexane (BSH),4(O-CH₂ CO-Gly-Gly-Cys.amide)-2-methylpropiophenone (ETAC);tris(acetamidoethyl)amine, bis-acetamidomethyl ether, bis-acetamidoethylether, α,ε-bis-acetyllysine, lysine and1,8-bis-acetamido-3,6-dioxa-octane, or derivatives thereof.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides receptor binding vasoactive intestinalpeptides and fragments, derivatives, analogues and mimetics thereof thatare useful as reagents in the preparation of receptor binding vasoactiveintestinal peptide radiopharmaceutical agents for diagnosis and therapy.

Embodiments of these receptor binding vasoactive intestinal peptidesprovided by this invention are receptor binding vasoactive intestinalpeptide reagents wherein the receptor binding vasoactive intestinalpeptides, fragments, derivatives, analogues and mimetics thereof arecovalently linked to a chelating moiety. Such receptor bindingvasoactive intestinal peptide reagents are capable of being radiolabeledto provide radiodiagnostic or radiotherapeutic agents. One example of aradiodiagnostic application using the radiolabeled agents of theinvention is scintigraphic imaging, wherein the location and extent ofVIP receptor-bearing tumors may be determined. The receptor bindingvasoactive intestinal peptide reagents of the invention can alsoadvantageously be radiolabeled with cytotoxic radioisotopes such asrhenium-186 or rhenium-188 for radiotherapeutic uses.

The term scintigraphic imaging agent as used herein is meant toencompass a radiolabeled agent capable of being detected with aradioactivity detecting means (including but not limited to agamma-camera or a scintillation detector probe).

Radiotherapeutic embodiments of the invention, on the other hand, areadvantageously labeled with rhenium-186 and rhenium-188. Suchembodiments are useful in the treatment of VIP-related diseases or otherailments in animals. Preferably humans, including but not limited tocolorectal cancer and other diseases characterized by the growth ofmalignant or benign rumors capable of binding VIP or VIP analogues viathe expression of VIP receptors on the cell surface of cells comprisingsuch tumors.

For the purposes of this invention, the term "VIP receptor bindingaffinity" is intended to mean binding affinity as measured by anymethods known to those of skill in the art, including, inter alia, thosemethods which measure binding affinity by a disassociation constant, aninhibition constant or an IC₅₀ value. The term "having an affinity of atleast one-tenth the affinity of radioiodinated VIP" is intended to meanthat the affinity is not less than ten times less than the affinity ofradioiodinated VIP, or that the inhibition constant (K_(i)) or IC₅₀ isnot more than 10 times that of radioiodinated VIP.

In the chelating moieties and receptor binding vasoactive intestinalpeptides covalently linked to such moieties that contain a thiolcovalently linked to a thiol protecting group {(pgp)^(S) } provided bythe invention, the thiol-protecting groups may be the same or differentand may be but are not limited to:

--CH₂ -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH-(aryl)₂, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--C-(aryl)₃, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH₂ -(4-methoxyphenyl);

--CH-(4-pyridyl)(phenyl)₂ ;

--C(CH₃)₃

-9-phenylfluorenyl;

--CH₂ NHCOR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ -NHCOOR (R is unsubstituted or substituted alkyl or aryl);

--CONHR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --S--CH₂ -phenyl

Preferred protecting groups have the formula --CH₂ --NHCOR wherein R isa lower alkyl having 1 and 8 carbon atoms, phenyl or phenyl-substitutedwith lower alkyl, hydroxyl, lower alkoxy, carboxy, or loweralkoxycarbonyl. The most preferred protecting group is anacetamidomethyl group.

For the purposes of this invention, the term "receptor bindingvasoactive intestinal peptide(s) is intended to encompassnaturally-occurring VIP, fragments, analogues, derivatives thereof thatspecifically bind to the VIP receptor expressed in a variety of celltypes recognized by those with skill in the art. Compounds designed tomimic the receptor-binding properties of VIP (i.e. mimetics) are alsoincluded in this definition and encompassed by the invention.

Particularly preferred embodiments of the reagents of the inventioninclude:

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(β-Dap)KCK.amide).amide

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCK.amide).amide

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(δ-Orn)GCK.amide).amide

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(BAT).amide.                  (SEQ ID NO.:2)

    HSDAVFTDNYTRLRKQMAVKKYLNSILNGGC.amide                      (SEQ ID NO.:3)

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO·GGCE·amide).mu    ltidot.amide and                                                                                                   - HSDAVFTDNYTRLRKQMAVKKYLNSILN(.epsil                                       on.-K)GC·amide                

All naturally-occurring amino acids are abbreviated using standardabbreviations (which can be found in G. Zubay, Biochemistry (2d. ed.),1988 (MacMillen Publishing: New York) p.33). For the purposes of thisinvention, the naturally-occurring amino acids are characterized aslipophilic (alanine, isoleucine, leucine, methionine, phenylalanine,tyrosine, proline, tryptophan and valine, as well as S-alkylatedderivatives of cysteine), hydrophilic (asparagine, glutamine, threonine,serine), acidic (glutamic acid and aspartic acid), basic (arginine,histidine and lysine). ε-K is intended to represent a covalent linkagevia the δ-amino group on the sidechain of a lysine residue. δ-Ornrepresents an ornithine residue in which the δ-amino group, rather thanthe typical α-amino group, is covalently linked to the carboxyl group ofthe adjacent amino acid to form a peptide bond, γ-Dab represents a2,4diaminobutyric acid residue in which the γ-amino group is covalentlylinked to the carboxyl group of the adjacent amino acid to form apeptide bond. β-Dap represents a 1,3-diaminopropionic acid residue inwhich the β-amino group is covalently linked to the carboxyl group ofthe adjacent amino acid to form a peptide bond. (BAT) represents N⁶,N⁹-bis(2-mercapto-2-methyl-propyl)-6,9-diazanonanoic acid; K.(BAT) andLys.(BAT) represent the amino acid lysine, acylated at the ε-amino groupon the amino acid sidechain to (BAT); C(BAT) and Cys(BAT) representS-(N⁶,N⁹ -bis(2-mercapto-2methylpropyl)-6,9-diazanonan-1-yl)cysteine;(BAM) is (N¹,N⁴ -bis(2-mercapto-2-methylpropyl)-1,4,10-triazadecane;(BAT-BM) is N-{2-(N',N'-bis(2-maleimidoethyl)aminoethyl)-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenyl-methylthiopropyl)-6,9-diazanonanamide;(BAT-BS) is N-{2-(N',N'-bis(2-succinimidoethyl)aminoethyl)-N⁶,N⁹-bis(2-mercapto-2-methylpropyl)-6,9-diazanonanamide; (BMME) isbis-maleimidomethylether; and (BSME) is bis-suεεinimidomethylether. Asused herein, the following amino acids and amino acid analogues areintended to be represented by the following abbreviations: Acm is thesulfhydryl protecting group acetamidomethyl; Pen is penicillamine; Acais 6-aminocaproic acid; Hly is homolysine; Apc isL-{S-(3-aminopropyl)cysteine; F_(D) is D-phenylalanine: W_(D) isD-tryptophan; Y_(D) is D-tyrosine; Cpa is L-(4-chlorophenyl)alanine: Thpis -amino-tetrahydrothiopyran carboxylic acid; D-Nal isD-2-naphthylalanine; Dpg is dipropylglycine; Nle is norleucine; Hcy ishomocysteine; Hhc is homohomocysteine; Aib is aminoisobutyric acid; Nalis 2-naphthylalanine, -Nal is D-2-naphthylalanine, Ain is2-aminoindan-2carboxylic acid; Achxa is 4-amino-cyclohexylalanine; Amfis 4-aminomethyl-phenylalanine; Aec isS-(2-aminoethyl)cysteine-aminopropyl)cysteine; APC isS-(3-aminopropyl)cysteine; Aes is O-(2-aminoethyl)serine; Aps is O-(³-aminopropyl)serine; Abu is 2-aminobutyric acid; and Nva is norvaline.

Receptor binding vasoactive intestinal peptides and derivatives andanalogues thereof provided by the present invention can be chemicallysynthesized in vitro. Peptides of the present invention can generallyadvantageously be prepared on a peptide synthesizer. The peptides ofthis invention are synthesized wherein the chelating moiety, or a uniqueattachment moiety for the chelating agent, is covalently linked to thepeptide during chemical synthesis in vitro, the chemical transformationsbeing well known to those with skill in the art. Such peptidescovalently-linked to the chelating moiety, or unique chelating agentattachment moiety, during synthesis are advantageous because specificsites of covalent linkage can be determined.

Chelating moieties of the invention are introduced into the targetreceptor binding vasoactive intestinal peptide, derivative or analogeither during peptide synthesis, or through attachment to a unique aminoacid which is incorporated into the vasoactive intestinal peptide duringpeptide synthesis. Thus cysteine or homocysteine may be incorporated ata specific position in the peptide during peptide synthesis and thechelating moiety can later be covalently attached site-specifically tothe cysteine or homocysteine thiol group. This invention provides forthe incorporation of chelating moieties in a site-selective fashion intovirtually any position in the peptide. The invention in particularprovides amino acid derivatives comprising radiolabel chelating moietieslinked to an amino acid sidechain, wherein either the chelator, or aunique amino acid to which the chelator may be subsequentlysite-specifically attached, is incorporated into the peptide during invitro peptide synthesis at a specific position in the peptide. Theinvention also provides peptides wherein the radiolabel chelatingmoieties are incorporated into the peptide at the carboxyl terminus. Inpreferred embodiments, the radiolabel chelating moiety is incorporatedinto or onto a sidechain of an amino acid of the synthetic, vasoactiveintestinal peptide, wherein the amino acid is a native or surrogateamino acid in the sequence

    -SDAVFTDNYTRLRKQMAVKKYLNSILN.amide                         (SEQ ID NO.:4)

of the peptide. In other preferred embodiments, the radiolabel chelatingmoiety is incorporated into the synthetic, vasoactive intestinal peptideat a site that is in the sidechain of an amino acid of the peptide,wherein the amino acid is a native or surrogate amino acid in thesequence

    -NYTRLRKQMAVKKYLNSILN.amide                                (SEQ ID NO.:5)

of the peptide. In other preferred embodiments, the radiolabel chelatingmoiety is incorporated into the synthetic, vasoactive intestinal peptideat a site that is in the sidechain of a native or surrogate amino acidof the peptide, wherein the amino acid is an amino acid in the sequence

    -KQMAVKKYLNSILN.amide                                      (SEQ ID NO.:6)

of the peptide.

In yet further preferred embodiments, the radiolabel chelating moiety isincorporated into the synthetic, vasoactive intestinal peptide at thecarboxyl terminus of the peptide.

It is a particular advantage of the reagents of the invention that theyare provided having the radiolabel chelating moiety, or a unique aminoacid to which the chelator may be subsequently site-specificallyattached, incorporated into the peptide during synthesis. This isadvantageous because it allows placement of the chelator at a knownposition in vasoactive intestinal peptide, or in the vasoactiveintestinal peptide fragment, analogue or derivative so as to avoiddecreasing the affinity of the peptide for the VIP receptor. Methods forintroducing chelators into peptides known in the prior art have beendeveloped predominantly from methods first developed for protein, which,being much larger than peptides, are not as sensitive to the effects ofnon-site-specific introduction of the chelating moiety. Peptidesproduced using prior art methods are disadvantageous as compared withthe site-specific introduction of the chelators in the peptides of thisinvention due to the likelihood of introducing the chelator in such away as to decrease peptide binding affinity, when using the methods ofthe prior art.

It is also an advantage of this invention that the peptides are providedas chemically-synthesized peptides. This is because chemical synthesisis a controlled process amenable to chemical engineering techniques thatare capable of providing a quality-controlled andpharmaceutically-suitable product. Chemical synthesis methods arepreferred over other methods, such as biological synthesis andextraction, which may involve the introduction of pathogens (viruses,mycoplasma, etc.) which require costly expenditures to remove or proveabsent. Products prepared by chemical synthesis are less expensive toproduce and more amenable to successful regulatory approval, therebyimpacting the ability to expeditiously bring pharmaceutical embodimentsinto the clinic and to market.

In forming a complex of radioactive technetium with the reagents of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the reagent in the presence of a reducingagent. Preferred reducing agents are dithionite, stannous and ferrousions; the most preferred reducing agent is stannous chloride. Means forpreparing such complexes are conveniently provided in a kit formcomprising a sealed vial containing a predetermined quantity of areagent of the invention to be labeled and a sufficient amount ofreducing agent to label the reagent with Tc-99m. Alternatively, thecomplex may be formed by reacting a reagent of this invention with apre-formed labile complex of technetium and another compound known as atransfer ligand. This process is known as ligand exchange and is wellknown to those skilled in the art. The labile complex may be formedusing such transfer ligands as tartrate, citrate, gluconate or mannitol,for example. Among the Tc-99m pertechnetate salts useful with thepresent invention are included the alkali metal salts such as the sodiumsalt, or ammonium salts or lower alkyl ammonium salts.

In a preferred embodiment of the invention, a kit for preparingtechnetium- or rhenium-labeled peptides is provided. An appropriateamount of the peptide reagent is introduced into a vial containing areducing agent, such as stannous chloride, in an amount sufficient tolabel the peptide with Tc-99m. Re-186 or Re-188. An appropriate amountof a transfer ligand as described (such as tartrate, citrate, gluconate,glucoheptanate or mannitol, for example) can also be included. The kitmay also contain conventional pharmaceutical adjunct materials such as,for example, pharmaceutically acceptable salts to adjust the osmoticpressure, buffers, preservatives and the like. The components of the kitmay be in liquid, frozen or dry form. In a preferred embodiment, kitcomponents are provided in lyophilized form.

Tc-99m, Re-186 and Re-188 labeled radiopharmaceuticals according to thepresent invention may be prepared by the addition of an appropriateamount of Tc-99m, Re-186 or Re-188, or radionuclide complexes thereof,into the vials and reaction under conditions described in Example 2hereinbelow.

Radioactively-labeled scintigraphic imaging agents provided by thepresent invention are provided having a suitable amount ofradioactivity. In forming Tc-99m radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 millicurie (mCi) to100 mCi per mL.

The imaging reagents provided by the present invention can be used forvisualizing sites of expression or hyperexpression of VIP receptors,including organs such as the colon for diagnosing disorders in theseorgans, and tumors, in particular gastrointestinal rumors, specificallycolorectal tumors that can be imaged. In accordance with this invention,the Tc-99m labeled peptide reagents are administered in a single unitinjectable dose. The Tc-99m labeled peptide reagents provided by theinvention may be administered intravenously in any conventional mediumfor intravenous injection such as an aqueous saline medium, or in bloodplasma medium. Generally, the unit dose to be administered has aradioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20mCi. The solution to be injected at unit dosage is from about 0.01 mL toabout 10 mL. After intravenous administration, imaging in vivo can takeplace in a matter of a few minutes. However, imaging can take place, ifdesired, in hours or even longer, after the radiolabeled peptide isinjected into a patient. In most instances, a sufficient amount of theadministered dose will accumulate in the area to be imaged within about0.1 of an hour to permit the taking of scintiphotos. Any conventionalmethod of scintigraphic imaging for diagnostic purposes can be utilizedin accordance with this invention.

This invention also provides peptides radiolabeled with cytotoxicradioisotopes such as rhenium-186 or rhenium-188 that may be used forradiotherapy of certain tumors as described above. For this purpose, anamount of radioactive isotope from about 10 mCi to about 2 mCi ma,y beadministered via any suitable clinical route, preferably by intravenousinjection.

This invention also provides VIP receptor-binding vasoactive intestinalpeptides covalently linked to a metal-binding moiety that are complexedwith a magnetic. paramagnetic, supermagnetic, or superparamagnetic metalatom, ion or particle, and methods for using such complexes formagnetic-based detection of localization of such receptor bindingvasoactive intestinal peptide complexes at tumor or other tissue sitesin vivo. Thus, the invention provides non-radioactive methods forlocalizing tumor and other vasoactive intestinal receptor expressingtissues in vivo.

This invention provides methods for using the diagnostic andradiodiagnostic and therapeutic and radiotherapeutic agents of theinvention. For radiolabeled embodiments of the agents of the invention,for example, Tc-99m labeled scintigraphic imaging agents, an effectivediagnostic or therapeutic amount of the diagnostic or radiodiagnostic ortherapeutic or radiotherapeutic agent of the invention are administered.In radiodiagnostic embodiments, localization of the radiolabel isdetected using conventional methodologies such as gamma scintigraphy. Innon-radioactive diagnostic embodiments. localization of sites ofaccumulation of the paramagnetic metal-labeled diagnostic agents of theinvention is achieved using magnetic resonance imaging methodologies.For the purposes of this invention, radiotherapy is defined as atherapeutic effect ranging from pain palliation to cure.

The imaging agents provided by the invention have utility for tumorimaging, particularly for imaging primary and metastatic neoplasticsites wherein said neoplastic cells express VIP receptors, and inparticular such primary and especially metastatic colorectal tumor cellsthat have been clinically recalcitrant to detection using conventionalmethodologies.

Those having skill in this art will recognize that efficaciousradiopharmaceuticals can be identified, tested and characterized usingany of a number of in vitro methodologies known in the art. Suchmethodologies include, inter alia, the determination of dissociationconstants or inhibition constants of binding of the radiopharmaceuticalsof the invention to their cognate VIP receptors, as well as comparisonof the affinity or avidity of such binding with binding of radiolabeled,for example, ¹²⁵ I-labeled. VIP itself, and in experiments wherein aradiopharmaceutical of the invention is used in competition withradiolabeled VIP, or in the converse experiments using unlabeled VIP incompetition with radiopharmaceutical of the invention.

In the practice of this invention, effective radiodiagnostic andradiotherapeutic agents are prepared as follows. Reagents of theinvention comprising receptor binding vasoactive intestinal peptides andvasoactive intestinal peptide fragments, analogues and derivativesthereof, are synthesized using the methods of the invention wherein thechelating moiety is incorporated into the peptide during chemicalsynthesis. The reagents of the invention are then complexed withrhenium, preferably as the ReO complex, as further disclosed herein. VIPreceptor binding is then evaluated in in vitro competition bindingassays as described herein using radioiodinated VIP, as disclosed inExample 4 below.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results, and areshown by way of illustration and not limitation.

EXAMPLE 1 Synthesis of BAT Chelators

A. Synthesis ofN-Boc-N'-(5-carboxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

a. 2-methyl-2-(triphenylmethylthio)propanal

Triphenylmethylmercaptan (362.94 g, 1.31 mol, 100 mol %) dissolved inanhydrous THF (tetrahydrafuran; 2 L) was cooled in an ice bath underargon. Sodium hydride (60% in oil: 54.39 g, 1.35 mol, 104 mol %) wasadded in portions over 20 min. 2-bromo-2-methylpropanal (206.06 g, 1.36mol, 104 mol %: see Stevens & Gillis. 1957. J. Amer. Chem. Soc. 79:3448-51) was then added slowly over 20 min. The reaction mixture wasallowed to warm to room temperature and stirred for 12 hours. Thereaction was quenched with water (1 L) and extracted with diethyl ether(3×1 L). The ether extracts were combined, washed with saturated NaClsolution (500 mL), dried over Na₂ SO₄ and filtered. The solvent wasremoved under reduced pressure to afford a thick orange oil. The crudeoil was dissolved in toluene (200 mL) and diluted to 2 L with hothexanes. The mixture was filtered through a sintered glass funnel andcooled at -5° C. for 12 hours. The white crystalline solid which formedwas removed by filtration to afford 266.36 g (59% yield) of the titlecompound. The melting point of the resulting compound was determined tobe 83-85° C. Nuclear magnetic resonance characterization experimentsyielded the following molecular signature:

¹ H NMR(300 MH_(z), CDCl₃): δ 1.24(s, 6H, 2CH₃), 7.2-7.35 (m, 9H),7.59-7.62 (m,6H), 8.69 (s, H, --COH).

¹³ C NMR (75 MH_(z), CDCl₃): δ 22.86, 55.66, 67.48, 126.85, 127.75,129.72, 144.79, 197.31.

b. N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

Ethylenediamine (1.3 mL, 0.0194 mol, 100 mol %) was added to2-methyl-2-(triphenylmethylthio)propanal (13.86 g, 0.0401 mol, 206 mol%) dissolved in methanol (40 mL) and anhydrous THF (40 mL) under argon,and the pH was adjusted to pH 6 by dropwise addition of acetic acid. Thesolution was stirred for 20 min at 20° C. Sodium cyanoborohydride (1.22g, 0.0194 mol, 100 mol %) was added and the reaction was stirred at roomtemperature for 3 hours. Additional sodium cyanoborohydride (1.08 g) wasadded and the reaction was stirred at 20° C. for 17 hours. A finalportion of sodium cyanoborohydride (1.02 g) was added and the reactionheated at reflux under argon for 6 hours. The reaction was quenched with0.5 M HCl (100 mL) and extracted with ethyl acetate (2×100 mL). Theorganic extracts were combined, sequentially washed with 2 M NaOH (60mL), saturated NaCl solution (60 mL), dried (Na₂ SO₄), and filtered. Thesolvent was removed under reduced pressure to give 16.67 g of crudeproduct which was crystallized from toluene/hexanes to afford 10.20 g(73% yield) of white crystals of the title compound. The melting pointof the resulting compound was determined to be 83-86° C. FABMS analysisyielded an m/z of 721 (MH+). Nuclear magnetic resonance characterizationexperiments yielded the following molecular signature:

¹ H NMR (300 MH_(z), CDCl₃): δ 1.12 (s, 12H, 4 CH₃), 1.64 (s, 4H, N--CH₂--C(Me)₂ --S), 2.52 (s, 4H, N--CH₂ --CH₂ --N), 5.31 (S, 2H, 2-NH).7.12-7.30 (m, 18H, Ar). 7.62-7.65 (m. 12H, Ar).

c.N-(5-carboethoxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

K₂ CO₃ (1.92 g, 13.9 mmol, 100 mol %) was added toN,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine (10.03 g,13.9 mmol) in CH₃ CN (60 mL), followed by ethyl 5-bromovalerate (3.30mL, 20.8 mmol, 150 mol %). The reaction was heated at reflux under argonovernight. The solution was then concentrated to a paste and partitionedbetween 0.25 M KOH (100 mL) and ethyl acetate (100 mL). The aqueouslayer was extracted with ethyl acetate (1×50 mL) and the combined ethylacetate layers were washed with 50 mL water and NaCl solution (2×50 mL),dried with Na₂ SO₄ and concentrated to an orange oil. Purification byflash chromatography (300 g flash silica, 100% CHCl₃ to 5% MeOH/CHCl₃)gave pure title compound (7.75 g, 66% yield). FABMS analysis yielded an(MH+) of 849 (compared with a calculated molecular weight of 849.24 forthe compound C₅₅ H₆₄ N₂ O₂ S₂).

d.N-Boc-N'-(5-carboxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine

1M KOH (25 mL, 25.0 mmol, 274 mol %) was added toN-(5-carboethoxypentyl)-N,N'-bis(2-methyl-2-triphenylmethylthiopropyl)ethylenediamine(7.75 g, 9.13 mmol) in dioxane (200 mL), followed by water (250 mL).Dioxane was then added dropwise with stirring until a homogeneoussolution was obtained. The reaction was heated at a slow refluxovernight. Most of the dioxane was removed by rotary evaporation and thepH of solution was adjusted to ˜7-8 with 1 M KH₂ PO₄ and saturatedNaHCO₃. The solution was then extracted with ethyl acetate (3×75 mL) andthe combined organic layers were washed with NaCl solution (50 mL),dried with Na₂ SO₄ and concentrated to a foam/solid (6.35 g, 85% yield).

To the crude product from the above reaction was added (BOC)₂ O (3.35 g,15.4 mmol, 200 mol %), CH₃ CN (50 mL) and methylene chloride (50 mL),followed by triethylamine (1.0 mL, 7.2 mmol, 93 mol %). The reaction wasstirred at room temperature under argon overnight. The reaction solutionwas then concentrated and partitioned between water (100 mL) and ethylacetate (50 mL). The aqueous layer was extracted with ethyl acetate(1×50 mL) and the combined ethyl acetate layers were washed with 5%citric acid and NaCl solution (50 mL each), then dried (Na₂ SO₄) andconcentrated to an orange oil. Purification by flash chromatography (200g flash silica, 100% CDCl₃ to 5% methanol/chloroform) gave pure titlecompound (2.58 g, 36% yield). FABMS analysis gave an (MH+) of 921(compared with the calculated value of 921.31 for the compound C₅₈ H₆₈N₂ O₄ S₂).

B. Synthesis ofN-Boc-N'-(5-carboxypentyl)-N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]ethylenediamine

a. N-N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]-ethylenediamine

A solution of N,N'-bis(2-mercapto-2-methylpropyl)ethylenediamine (11.23g, 47.5 mmol; see, DiZio et al., 1991, Bioconjugate Chem 2: 353 andCorbin et al., 1976. J. Org. Chem. 41: 489) in methanol (500 mL) wascooled in ice/water bath and then saturated with gaseous ammonia over 45min. To this was added 4-methoxybenzyl chloride (17.0 mL, 125 mmol, 264mol %). The reaction was allowed to warm to room temperature overnightwith stirring under argon. The solution was concentrated to a paste andthen partitioned between diethyl ether (150 mL) and 0.5 M KOH (200 mL).The aqueous layer was further extracted with diethyl ether (2×50 mL).The combined organic layers were washed with NaCl solution andconcentrated to a clear colorless oil. The oil dissolved in diethylether (200 mL) and then acidified with 4.0 M HCl in dioxane until nofurther precipitation was seen. The white precipitate was collected byfiltration and washed with diethyl ether. The white solid wasrecrystallized from hot water at a pH of ˜2. The product was collectedby filtration to afford 29.94 g as a mix of mono- and di-HCl salts. TheHCl salts were partitioned between 1M KOH (100 mL) and ethyl acetate(100 mL). The aqueous was extracted with ethyl acetate (2×30 mL) and thecombined organic layers were washed with NaCl solution, dried with Na₂SO₄ and concentrated to give pure product as the free base as a lightyellow oil (18.53 g, 82% yield). Nuclear magnetic resonancecharacterization experiments yielded the following molecular signature:

¹ H NMR (300 MHz, CDCL₃): δ 7.25 (d, 4H, J=9), 6.83 (d, 4H, J=9), 3.78(s, 6H), 3.67 (s, 4H), 2.63 (s, 4H), 2.56 (s, 4H), 1.34 (s, 12H).

b.N-(5-carboethoxypentyl)-N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]ethylenediamine

To N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]-ethylenediamine(4.13 g, 8.66 mmol) in CH₃ CN (50 mL) was added K₂ CO₃ (1.21 g, 8.75mmol, 101 mol %) followed by ethyl 5-bromovalerate (2.80 mL, 17.7 mmol,204 mol %). The reaction was stirred at reflux overnight and was thenconcentrated to a paste in vacuo. The residue was partitioned betweenethyl acetate (100 mL) and 0.5 M KOH (100 mL). The aqueous layer wasextracted with ethyl acetate (1×50 mL) and the combined organic layerswere washed with NaCl solution (50 mL), dried with Na₂ SO₄ andconcentrated to a yellow oil (˜6 g). Purification by normal-phasepreparative HPLC (100% CHCl₃ to 5% methanol/chloroform over 25 min.)afforded pure title compound (1.759 g, 34% yield). FABMS analysis gavean (MH+) of 605 (compared with the value of 604.90 calculated for C₃₃H₅₂ N₂ O₄ S₂). Nuclear magnetic resonance characterization experimentsyielded the following molecular signature:

¹ H NMR (300 mH_(z), CDCl₃): δ 7.25 (d, 4H, J=8.5), 6.83 (d, 4H, J=8.5),4.13 (q, 2H, J=7), 3.793 (s, 3H), 3.789 (s, 3H), 3.74 (s, 2H), 3.67 (s,2H), 2.6 (m, 10H), 2.31 (t, 2H, J=7), 1.6 (m, 2H), 1.5 (m 2H), 1.34 (s12H), 1.28 (t, 3H, J=7).

c.N-Boc-N'-(5-carboxypentyl)-N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]ethylenediamine

ToN-(5-carboethoxypentyl)-N,N'-bis-[2-(4-methoxybenzylthio)-2-methylpropyl]ethylenediamine(586 mg, 0.969 mmol) in THF (40 mL) was added water (30 mL) and 1 M KOH(2.5 mL, 2.5 mmol, 260 mol %). The homogeneous solution was heated to aslow reflux overnight. The solution was then cooled to room temperatureand the THF was removed under rotary evaporation. The residue wasdiluted to 50 mL with H₂ O and the pH was adjusted to ˜2-3 with 1 M HCl.The solution was extracted with ethyl acetate (3×30 mL) and the combinedorganic layers were washed with NaCl solution (50 mL), dried with Na₂SO₄ and concentrated to give crude acid (422 mg, 75% yield).

To the crude product from the above reaction was added CH₃ CN (40 mL)and (BOC)₂ O (240 mg, 1.10 mmol, 150 mol %) followed by triethylamine(0.200 mL, 1.43 mmol, 196 mol %). The homogenous solution stirred atroom temperature overnight under argon. The solution was thenconcentrated to a paste and partitioned between ethyl acetate (25 mL)and 1 M KH2PO₄ (25 mL). The organic layer was washed with 5% citric acid(2×5 mL) and NaCl solution (25 mL), dried with Na₂ SO₄ and concentratedto a yellow oil. Purification by flash chromatography (50 mL flashsilica gel, 100% chloroform to 15% methanol/chloroform) gave pure titlecompound (344 mg, 70% yield). FABMS analysis gave an (MH+) of 677(compared to the value of 676.97 calculated for the compound C₃₆ H₅₆ N₂O₆ S₂). Nuclear magnetic resonance characterization experiments yieldedthe following molecular signature: ¹ H NMR (300 MHz, CDCl₃): δ 7.20 (d,4H, J=7), 6.79 (d, 4H, J=7), 3.75 (S, 3H), 3.74 (S, 3H), 3.68 (M, 4H),3.35 (M, 4H), 2.65 (M, 2H), 2.53 (M, 4H), 2.31 (M, 2H), 1.59 (M, 2H),1.43 (S, 11H), 1.30 (S, 6H), 1.26 (S, 6H).

C. Synthesis ofBAT-BM(N-[2-(N,N'-bis(2-maleimidoethyl)aminoethyl)]-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide)

BAT-BM was prepared as follows. BAT acid (N⁹ -(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanoic acid)(10.03 g, 10.89 mmol) and 75 mL of dry methylene chloride (CH₂ Cl₂) wereadded to a 250 mL round-bottomed flask equipped with magnetic stir barand argon balloon. To this solution was added diisopropylcarbodiimide(3.40 mL, 21.7 mmol, 199 mole %), followed by N-hydroxysuccinimide(3.12g, 27.1 mmol, 249 mole %). This solution was observed to becomecloudy within 1 h, and was further incubated with stirring for a totalof 4 h at room temperature. A solution of tris(2-aminoethyl)amine (30mL, 200 mmol, 1840 mole %) in 30 mL methylene chloride was then addedand stirring continued overnight. The reaction mixture was thenconcentrated under reduced pressure, and the residue partitioned betweenethylacetate (150 mL) and 0.5M potassium carbonate (K₂ CO₃ ; 150 mL).The organic layer was separated, washed with brine and concentrated togive the crude product N-[2-N',N'-bis(2-aminoethyl)aminoethyl)]-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide as afoam/oil.

This crude product was added to a 1000 mL round-bottomed flask, equippedwith magnetic stir bar, containing 300 mL THF, and then 30 mL saturatedsodium bicarbonate (NaHCO₃), 100 mL water and N-methoxycarbonylmaleimide(6.13 g, 39.5 mmol, 363 mole %) were added. This heterogeneous mixturewas stirred overnight at room temperature. THF was removed from themixture by rotary evaporation, and the aqueous residue was twiceextracted with ethylacetate (2×75 mL). The combined organic layers ofthese extractions were washed with brine, dried over sodium sulfate,filtered through a medium frit and concentrated to about 12 g of crudeproduct. Purification by liquid chromatography (250 g silicondioxide/eluted with a gradient of chloroform→2% methanol in chloroform)afforded 5.3 g of pure product(N-[2-(N',N'-bis(2-maleimidoethyl)aminoethyl)]-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide)(equivalent to 40% yield), along with approximately 5 g of crude productthat can be re-purified to yield pure product. Chemical analysis of thepurified product confirmed its identity as BAT-BM as follows:

¹ H NMR (200 mHz, CDCl₃): δ 0.91 (12H,s), 1.38 (9H,s), 1.2-1.6 (4H,m),2.06 (2H,s), 2.18(2H,t,J=7), 2.31 (4H,m), 2.55 (2H,t,J=5), 2.61(4H,t,J=6), 2.99 (2H,s), 3.0-3.3 (4H,m), 3.46 (4H,t,J=6), 6.49(--NH,t,J=4), 6.64 (4H,s), 7.1-7.3 (18H,m), 7.6 (12H,t,J =17).

D. Synthesis of [BAT]-conjugated(eN) Lys(aN-Fmoc) [N-e(N⁹-t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonanoyl)-N-a-Fmoc-lysine

A 100 mL single-necked round-bottomed flask, equipped with stir bar andargon balloon, was charged with N⁹ -(t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonanoic acid (BATacid: 3.29 g, 3.57 mmol) in 50 mL CH₂ Cl₂ at room temperature. To thiswas added diisopropylcarbodiimide (DIC: 580 μL, 3.70 mmol, 104 mole %)followed immediately by N-hydroxysuccinimide (HOSu: 432 mg, 3.75 mmol,105 mole %). The reaction was stirred overnight at room temperatureduring which time a white precipitate developed. The mixture wasfiltered and the filtrate concentrated to a solid foam. The crude foam,in a 100 mL round-bottomed flask, was dissolved in 75 mL of a 2:1mixture of dimethoxyethane and water. To this homogeneous solution wasadded N-a-Fmoc-lysine hydrochloride (1.52 g, 3.75 mmol, 105 mole %)followed by K₂ CO₃ (517mg, 3.74 mmol, 105 mole %), and the yellowsolution stirred overnight at room temperature. The solution was thenpoured into a 250 mL erlenmeyer flask containing 100 mL of ethyl acetateand 100 mL of water. The organic layer was separated and the aqueouslayer further extracted with 50 mL ethyl acetate. The combined organiclayers were washed once with brine (100 mL), dried over Na₂ SO₄ andconcentrated to a yellow solid. This crude product was purified bylow-pressure liquid chromatography (150 g SiO₂, eluted with CHCl₃ ->10%methanol in CHCl₃). In this way, 3.12 g of the named compound wasprepared (69% yield). Chemical analysis of the purified productconfirmed its identity as follows:

¹ H NMR (300 MHz, CDCl₃): δ 0.88 (12H,s,broad), 1.05-1.45 (19H,m),1.8-2.1 (4H,m), 1.8-2.47 (4H,m), 2.75-3.2 (6H,m), 3.9-4.3 (4H,m), 7.2(22H,m), 7.6 (16H,m), FABMS MH⁺ was predicted to be 1270.6 and found tobe 1272.

E. Synthesis of BAM (N¹ -(t-butoxycarbonyl)-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4,10triazadecane

A 250 mL single-necked round-bottomed flask, equipped with a stir bar,reflux condenser and argon balloon, was charged with N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-ethylenediamine (BAT-I;10.0 g, 14.01 mmol) in 50 mL of CH₃ CN and 30 mL dioxane. To this wasadded N-(5-bromopentyl)-phthalimide (8.04 g, 27.1 mmol, 194 mole %)followed by K₂ CO₃ (2.95 g, 21.3 mmol, 152 mole %). The mixture washeated at reflux under argon for two days. The reaction mixture was thenconcentrated and the residue partitioned between 150 mL water and 150 mLethyl acetate. The organic layer was separated and the aqueous layer (atpH of about 10) was further extracted with 50 mL ethyl acetate. Thecombined organic layers were washed once with brine (75mL), dried overNa₂ CO₃ and concentrated to an oil. Purification by low-pressure liquidchromatography (300 g SiO₂, CHCl₃ ->2% methanol in CHCl₃) afforded 9.20g of 9-phthalimido-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4-diazanonane as a yellowfoam (70% yield). Chemical analysis of the purified product of thisintermediate confirmed its identity as follows:

¹ H NMR (300 MHz, CDCl₃): δ 1.01 (6H,s), 1.03 (6H,s), 1.15-1.4 (2H,t),1.98 (2H,s), 2.10 (2H,s), 2.28 (2H,m), 2.45 (3H,m), 3.68 (2H,t),7.15-7.35 (18H, m), 7.62 (12H, t), 7.72 (2H, m), 7.85 (2H,m), FABMS MH⁺was predicted to be 935.4 and found to be 936.

A 500 mL single-necked round-bottomed flask, equipped with stir bar, wascharged with 9-phthalimido-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4-diazanonane (8.83 g,9.43 mmol) in 75 mL of CH₃ CN and 20 mL CH₂ Cl₂. To this was added K₂CO₃ (1.30 g, 9.41 mmol, 100 mole %), followed by di-tert-butyldicarbonate (2.15 g, 9.85 mmol, 104 mole %), and the reaction stirred atroom temperature overnight. The reaction mixture was then concentratedand partitioned between 100 mL each of water and ethyl acetate. Theorganic layer was separated and the aqueous layer was further extractedwith 50 mL ethyl acetate. The combined organic layers were washed oncewith brine (75 mL), dried over Na₂ SO₄ and concentrated to give 9.69 gof crude 9-phthalimido-N¹ -(t-butoxycarbonyl)-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4-diazanonane as a yellowfoam (99% crude yield). This crude product was used without furtherpurification.

A 250 mL single-necked round-bottomed flask, equipped with stir bar andreflux condenser, was charged with 9-phthalimido-N¹-(t-butoxycarbonyl)-N¹ N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4diazanonane (5.50 g,5.319.43 mmol) in 25 mL of THF. To this was added 100 mL ethanol and 5mL water. The addition of water caused the starting material toprecipitate out of solution. Hydrazine hydrate (1.2 mL, 24.7 mmol, 466mole %) was added, and the reaction heated at reflux for two days. Thereaction mixture was concentrated and partitioned between 100 mL each ofwater and 0.25M K₂ CO₃. The organic layer was separated and washed oncewith brine (75 mL), dried over Na₂ SO₄ and concentrated to a solid foam.Purification of the crude product by low-pressure liquid chromatography(100 g SiO₂, CHCl₃ ->5% methanol in CHCl₃, the column pre-treated with200 mL 2% triethylamine in CHCl₃) afforded 3.27 g of pure N¹-(t-butoxycarbonyl)-N¹,N⁴-bis[2-methyl-2-(triphenylmethylthio)propyl]-1,4,10-triazadecane as ayellow foam (68% yield). Chemical analysis of the purified productconfirmed its identity as follows:

¹ H NMR (300 MHz, CDCl₃): δ 0.9 (12H,s), 1.2 (6H,s), 1.36 (9H,s), 2.05(4H,m), 2.24 (2H,t), 2.31 (2H,t), 2.62 (3H,t), 3.0 (2H,s,broad), 3.1(2H,s,broad), 7.2 (18H,m), 7.6 (12H,t ). FABMS MH⁺ was predicted to be905.5 and found to be 906.5.

F. Synthesis of [BAT]-conjugated(S)Cys(aN-Fmoc)(N(Fmoc)-S-(N⁹-t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonane-1-yl)cysteine)

a. Synthesis of (N⁹ -t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonan-1-ol(BAT-pentanol)

To a 500 mL round-bottomed flask containing BAT acid (10.4 g, 10.89mmol) in 250 mL dry THF under argon was added 12.1 mL of 1M BH₃ -THF(121 mmol), the addition being performed slowly over 5 min. Uponaddition, foaming occurred. This reaction mixture was incubated withstirring at room temperature overnight. The reaction was quenched withexcess methanol (50 mL) and incubated with stirring for an additional 2h. The solution was concentrated by evaporation and the residuepartitioned between 200 mL ethyl acetate, 100 mL 1M citric acid and 100mL 1M KH₂ PO₄. The organic layer was isolated and washed sequentiallywith 100 mL each of water, saturated NaHCO₃ and brine. The washedorganic layer was then dried over anhydrous Na₂ SO₄ and concentrated byevaporation to a white foam to give 8.82 g of crude product (89% yield).Chemical analysis of the purified product confirmed its identity asfollows:

¹ H NMR (300 MHz, CDCl₃): δ 0.91 (12H,s), 1.2-1.3 (4H,m), 1.38 (9H,s),1.4-1.6 (4H,m), 2.08 (1H,s), 2.2-2.4 (4H,m), 2.95-3.05 (2H,br),3.05-3.25 (2H,br), 3.58 (2H,t,J=6), 7.12-7.28 (18H,m), 7.61 (12H,t,J=7).

b. Synthesis of O-methanesulfonyl-(N⁹ -t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonan-1-ol(BAT-mesylate)

To a 500 mL round-bottomed flask containing BAT-pentanol (8.80 g, 9.70mmol) in 100 mL dry THF under argon was added 1.6 mL triethylamine (11.5mmol) followed by 0.83 mL methanesulfonyl chloride (10.7 mmol) withstirring. The solution became cloudy white within a few minutes of theseadditions. Stirring was continued overnight at room temperature. Thesolution was concentrated by evaporation and then partitioned between100 mL ethylacetate and 100 mL 1M KH₂ PO₄. The ethyl acetate layer wasisolated and washed with 100 mL brine, and then dried over anhydrous Na₂SO₄ and evaporated to give 9.61 g of crude product (100% yield) as alight yellow foam.

c. Synthesis of S-(N⁹ -t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonan-1-yl)cysteine(H-Cys(BAT)-OH)

In a 20 mL scintillation vial was combined cysteine (1.3 g. 10.7 mmol),4 mL 5.4M sodium methoxide (21.6 mmol) and 5 mL anhydrous methanol. Thishomogenous solution was added to a solution of BAT-mesylate (9.57 g.9.71 mmol) in 80 mL anhydrous dimethylformamide (DMF) and stirredovernight at room temperature under argon. The solvents were removed byrotary evaporation under reduced pressure and the residue partitionedbetween 100 mL ethyl acetate and 100 mL 1M KH₂ PO₄. The ethyl acetatelayer was isolated and washed sequentially with 100 mL each of saturatedNaHCO₃ and brine, and then dried over anhydrous Na₂ SO₄ and concentratedby evaporation to yield a white foam. This product was notcharacterized, but was used immediately in the following syntheticreaction.

d. Synthesis of N-fluorenylmethoxycarbonyl-S-(N⁹-t-butoxycarbonyl)-N⁶,N⁹-bis[2-methyl-2-(triphenylmethylthio)propyl]-6,9-diazanonan-1-yl)cysteine(Fmoc-Cys(BAT)-OH)

The crude H-Cys(BAT)-OH prepared as described above was assumed toconstitute 9.71 mmol product. The entire amount of this crude productwas dissolved in 200 mL THF and 150 mL water in a 1 Erlenmeyer flask. Tothis homogeneous solution was added 1.34 g K₂ CO₃ (9.72 mmol) followedby 3.28 g N-fluorenylmethoxycarbonyloxysuccinimide (9.73 mmol). Thisreaction mixture was stirred overnight at room temperature. Most of theTHF was then removed from the reaction mixture by rotary evaporation,and the remainder partitioned between 100 mL ethylacetate and 100 mL 1MKH₂ PO₄. The ethylacetate layer was isolated and washed with 50 mLbrine, dried over anhydrous Na₂ SO₄ and concentrated to yield a yellowfoam. This crude product was purified by liquid chromatography, using300 g silica gel and a linear gradient of 0-3% methanol in chloroform.Chromatography yielded 10.1 g pure Fmoc-Cys(BAT)-OH (84% yield fromBAT-mesylate). Chemical analysis of the purified product confirmed itsidentity as follows:

¹ H NMR (300 MHz, CDCl₃): δ 0.85 (12H,s), 1.23 (4H,br), 1.35 (9H,s), 1.5(2H,m), 2.0-2.15 (2H,m), 2.15-2.35 (4H,m), 2.51 (2H,t,J=6), 2.85-3.05(4H,m), 3.15 (2H,br), 4.1-4.2 (1H,m), 4.2-4.4 (3H,m), 7.05-7.4 (22H,m),7.5-7.65 (14H,m), 7.71 (2H,d,J=7).

EXAMPLE 2 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodimide/hydroxybenzotriazole or2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethyl henoxymethyl-polystyrene (HMP) resin or Sasrin orchiorotrityl resin for carboxyl-terminus acids or Rink amide resin forcarboxyl-terminus amides.

Where appropriate, Fmoc-Cys(BAT) and Nα-Fmoc-Nε-(BAT)Lys weresynthesized as described in Example 1.

Where appropriate, 2-chloroacetyl,2-bromoacetyl and2-bromo-3-phenylpropionyl groups are introduced either by using theappropriate 2-halo acid as the last residue coupled during SPPS, or bytreating the N-terminus free amino acid peptide bound to the resin witheither 2-halo acid/diisopropylcarbodiimide/N-hydroxysuccinimide/NMP or2-halo acid anhydride/diisopropylethylamine/NMP.

Where appropriate, HPLC-purified 2-haloacylated peptides are cyclized bystirring an 0.1-1.0 mg/mL solution in phosphate or bicarbonate buffer ordilute ammonium hydroxide (pH 8.0), optionally containing 0.5-1.0 mMEDTA, or acetonitrile or THF for 1-48 h followed optionally byacidification with acetic acid, lyophilization and HPLC purification.

Where appropriate, thiol-containing peptides are reacted withchloroacetyl-containing thiol-protected Tc-99m complexing moieties at pH10 for 0.5-4 hours at room temperature, followed by acetic acidacidification and evaporation of the solution to give the correspondingpeptide-sulfide adduct. Deprotection and purification are routinelyperformed as described to yield the chelator-peptide conjugate.

Where appropriate, BSME adducts are prepared by reacting singlethiol-containing peptides (5 to 50 mg/mL in DMF buffered to pH 7 withN-methylmorpholine or N-ethyl-morpholine, or 50 mM sodium phosphatebuffer, pH 7-8, optionally containing 0.5 mM EDTA or DMF or THF oracetonitrile) with 0.5 molar equivalents of BMME(bis-maleimidomethylether) pre-dissolved in acetonitrile at roomtemperature for approximately 1-18 hours. The solution was concentratedand the product was purified by HPLC.

Where appropriate, TSEA adducts are prepared by reacting singlethiol-containing peptide (at concentrations of 10 to 100 mg/mL peptidein DMF buffered to pH 7 with N-methylmorpholine or N-ethylmorpholine or5 to 50 mg/mL peptide in 50 mM sodium phosphate, pH 7-8, optionallycontaining 0.5 mM EDTA or DMF or THF or acetonitrile) with 0.33 molarequivalents of TMEA (tris(2-maleimidoethyl)amine) pre-dissolved inacetonitrile or DMF, with or without 1 molar equivalent oftriethanolamine, at room temperature for approximately 1-18 h. Suchreaction mixtures containing adducts are concentrated and the adductsare then purified using HPLC.

Where appropriate, (BAM) (N¹,N⁴-bis(2-mercapto-2-methylpropyl)-1,4,10-triazadecane) is conjugated tothe peptide by first activating the peptide carboxylate with a mixtureof diisopropylcarbodiimide/N-hydroxysuccinimide or HBTU/HOBt in DMF, NMPor methylene chloride, followed by coupling in the presence ofdiisopropylethylamine. After coupling, the conjugates are deprotected asdescribed above.

Where appropriate, (BAT) (N⁶,N⁹-bis(2-mercapto-2-methylpropyl)-6,9-diazanonanoic acid) is incorporatedinto peptides as protected amino acid derivatives, such as(Nα(Fmoc)-Nε(N-Boc)-S,S'-bistrityl-BAT)lysine (prepared fromNα(Fmoc)-lysine and Nε(N-Boc)-S,S'-bistrityl-BAT, or as(N(Fmoc)-S,S'-bistrityl-BAT)cysteine (prepared as described in Example1F, above) during peptide synthesis and then deprotected after cleavageof the completed peptide from the synthetic resin.

Where appropriate, BAT-BS(N-{2-(N',N'-bis(2-succinimidoethyl)aminoethyl)}-N⁶,N⁹-bis(2-methyl-2-mercaptopropyl)-6,9-diazanonanamide) adducts areprepared by reacting single thiol-containing peptide (at concentrationsof 2 to 50 mg/mL peptide in DMF buffered to pH 7 with N-methylmorpholineor N-ethylmorpholine, or in 50 mM sodium phosphate (pH 7-8), optionallycontaining 0.5 mM EDTA or DMF or THF or acetonitrile) with 0.5 molarequivalents of BAT-BM (N-{2-(N',N'-bis(2-maleimidoethyl)aminoethyl)}-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide)pre-dissolved in acetonitrile or THF, at room temperature forapproximately 1-18 h. The solution is then evaporated to dryness and(BAT-BS)-peptide conjugates deprotected by treatment with 10 mL TFA and0.2 mL triethylsilane for 1 h. The solution is concentrated, the productadducts precipitated with ether, and then purified by HPLC.

Where appropriate, peptide precursors are cyclized (between the amino-and carboxyl-termini) by reaction of the sidechain-protected, N-terminalfree amine and C-terminal free acid with diphenylphosphorylazide.

Sasrin resin-bound peptides are cleaved using a solution of 1% TFA indichloromethane to yield the protected peptide. Where appropriate,protected peptide precursors are cyclized between the amino andcarboxyl-termini by reaction of sidechain-protected, terminal free amineand carboxyl-terminal free acid using diphenylphosphorylazide.

HMP or Rink amide resin-bound products are routinely cleaved andprotected cyclized peptides deprotected using a solution comprised oftrifluoroacetic acid (TFA), or TFA and methylene chloride, optionallycomprising water, thioanisole, ethanedithiol, and triethylsilane ortriisopropylsilane in ratios of 100:5:5:2.5:2, for 0.5-3 hours at roomtemperature. Where appropriate, products were re-S-tritylated intriphenolmethanol/TFA, and N-Boc groups re-introduced into the peptideusing (Boc)₂ O.

Crude peptides are purified by preparative high pressure liquidchromatography (HPLC) using a Waters Delta Pak C18 column and gradientelution using 0.1% trifluoroacetic acid (TFA) in water modified withacetonitrile. Acetonitrile is evaporated from the eluted fractions whichare then lyophilized. The identity of each product is confirmed by fastatom bombardment mass spectroscopy (FABMS) or by electrospray massspectroscopy (ESMS).

Receptor binding vasoactive intestinal peptides, derivatives andanalogues synthesized as provided herein, as well as the products ofsuch synthesis identified by FABMS, are shown in Table I below.

                                      TABLE I                                     __________________________________________________________________________    Code                                                                              Peptide                            M.sup.+  (ESMS)                        __________________________________________________________________________    P887                                                                              HSDAVFTDNYTRLRKQMAVKKYLNSILN(e-K)GC.amide                                                                        3614                                     P916 HSDAVFTDNYTRLRKQMAVKKYLNSILNGGC.amide 3557                               P917 HSDAVFTDNYTRLRKQMAVKKYLNSILNC(BAT).amide 3734                            P1085 HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(β-Dap)KCK.amide).a                                           mide 3932                                P1087 HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCK.amide).amide 3832                                              P1086 HSDAVFTDNYTRLRKQMAVKKYLNSILN                                           C(CH.sub.2 CO.(δOrn)GCK.amide                                           ).amide 3888                             P1088 HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCE.amide).amide           __________________________________________________________________________                                           3832                                    ESMS is electrospray mass spectrometry                                   

EXAMPLE 3 A General Method for Radiolabeling with Tc-99m

0.1 mg of a peptide prepared as in Example 2 was dissolved in 0.1 mL or0.2 mL of water or 0.9% saline. Tc-99m gluceptate was prepared byreconstituting a Glucoscan vial (E.I. DuPont de Nemours, Inc.,Wilmington, Del.) with 0.25 mL of Tc-99m sodium pertechnetate containingup to 200 mCi and allowed to stand for 15 minutes at room temperature.25 μl of Tc-99m gluceptate was then added to the peptide and thereaction allowed to proceed at room temperature or at 100° C. for 15 or55 min and then filtered through a 0.2 μm filter.

The Tc-99m labeled peptide purity was determined by reverse-phase HPLCusing the following conditions: a Waters Delta Pak C-18. 5μ, 3.9 mm×150mm analytical column was loaded with each radiolabeled peptide, and thepeptides eluted at a solvent flow rate equal to 1 mL/min (Delta-Pak).Gradient elution was performed using a gradient of 20-50% SolventB/Solvent A (Solvent A is 0.1% CF₃ COOH in water and Solvent B is 0.1%CF₃ COOH in 90/10 CH₃ CN/H₂ O) for 20 min., followed by 100% B/A for 3min.

Radioactive components were detected using an in-line radiometricdetector linked to an integrating recorder. Tc-99m gluceptate and Tc-99msodium pertechnetate elute between 1 and 4 minutes under theseconditions, whereas the Tc-99m labeled peptides eluted after a muchgreater amount of time. Peptides were detected by in-linespectrophotometric detection at 220 nm.

Non-radioactive rhenium complexes were prepared by co-dissolving each ofthe peptide reagents of the invention with about one molar equivalent oftetrabutylammonium oxotetrabromorhenate (+5), prepared as described byCotton et al. (1966. Inorg. Chem. 5: 9-16) in dimethylformamide oracetonitrile/water and stirred for 0.5-5 days. The rhenium complexeswere isolated by reverse phase HPLC as described above for Tc-99mlabeled peptides and were characterized by FABMS or ESMS.Non-radioactive peptides were detected as peptides by in-linespectrophotometric detection at 220 nm.

Radioactive rhenium complexes, using for example Re-186 or Re-188, areprepared from the appropriate perrhenate salts using the same protocolas for Tc-99m labeling, or by adding a reducing agent to a solution ofthe peptide and perrhenate, or optionally using a ligand transfer agentsuch as citrate and incubating the reaction at a temperature betweenroom temperature and 100° C. for between 5 and 60 min.

Results of HPLC purification of peptides. Tc-99m labeled peptides andReO-complexed peptides are shown in Table II.

                  TABLE II                                                        ______________________________________                                                   HPLC Retention Time (min.)                                         Peptide      Peptide  ReO-complex                                                                              Tc-99m Labeled                               ______________________________________                                        (VIP)-(εK)GC.amide                                                                 11.6     12.7       12.9                                           (VIP)-GGC.amide 12.1 12.9 12.8                                                (VIP)-C(BAT).amide 13.8 17.9 18.4                                           ______________________________________                                         where VIP = HSDAVFTDNYTRLRKQMAVKKYLNSILN                                 

EXAMPLE 4 Biological Assays

Peptides of the invention, or ReO-complexed embodiments thereof, wereassayed for biological activity in competition binding assays with ¹²⁵I-labeled VIP.

Such assays are performed in a standard assay of VIP binding, usingmembranes isolated from guinea pig lung; on peripheral blood monocytesand platelets; and on a variety of human tumor cell lines.

In the practice of these methods, VIP was radioiodinated using theiodogen method, as described in Schanen et al. (1991, Lancet 6:395-396). Briefly, 50 μg VIP in 10 μL 0.5M phosphate buffer (pH 7.5), anappropriate amount of the radioisotope, and 6 μg iodogen were incubatedto room temperature for about 30 min with gentle stirring.Radioiodinated VIP was purified from unincorporated radioiodine by HPLCchromatography, and dissolved in phosphate buffered saline (PBS)supplemented with 0.1% human serum albumin and 0.1% Tween-80 detergent.

In assays using isolated rat guinea pig lung membranes, lungs wereobtained from male guinea pigs and membranes isolated essentially asdescribed in Carstairs and Barnes (1986, J. Pharmacol. Exp. Ther. 239:249-255, incorporated by reference). 0.2 mg of membrane preparation wasincubated with 0.01 nM ¹²⁵ I-labeled VIP in the presence or absence ofvarying concentrations of the peptides of the invention or ReO complexesthereof. From a comparison of the extent of binding in the presence orabsence of the unlabeled VIP compounds, a concentration was determinedfor each compound corresponding to inhibition of ¹²⁵ I-labeled VIPbinding by 50% (termed the IC₅₀).

Using these procedures, results for each of the tested compounds areshown in Table IV. These results indicate that the peptides and ReOcomplexes of the peptide of the invention are potent inhibitors of VIPbinding to VIP receptor-expressing lung membranes.

                  TABLE IV                                                        ______________________________________                                        VASOACTIVE INTESTINAL PEPTIDE                                                   (VIP) RECEPTOR BINDING COMPOUNDS                                                                                  Ki (nM)                                     Ki of Re                                                                    Code Sequence (nM) complex                                                  ______________________________________                                        P1085 {VIP}C.amide           5.1    5.0                                          (CH.sub.2 CO.(β-Dap)KCK.amide)                                          P900 VIP (SEQ ID NO: 1) 5.1* --                                               P1087 {VIP}C.amide (CH.sub.2 CO.GGCK.amide) 10 6.0                            P1086 {VIP}C.amide (CH.sub.2 CO.(δ-O)GCK.amide) 3.7 6.3                 P917 {VIP}C(BAT).amide (SEQ ID NO: 2) 7.8 68                                  P916 {VIP}GGC.amide (SEQ ID NO: 3) 24 8.6                                     P1088 {VIP}C.amide (CH.sub.2 CO.GGCE.amide) 16 17                             P887 {VIP}(ε-K)GC.amide 6.5 20                                      ______________________________________                                         *VIP = HSDAVFTDNYTRLRKQMAVKKYLNSILN.amide (SEQ ID NO: 1) tested blind (as     P900)                                                                    

Similar experiments were performed using normal peripheral humanplatelets and mononuclear cells.

In these experiments, platelets are isolated according to the method ofVirgolini et al. (1990, Brit. J. Cancer 12: 849-861). Peripheralmononuclear cells (PMNCs) are isolated from whole blood buffy coat byFicoll density gradient centrifugation (p=1.077). Before assay, cellsare washed in 50 mM Tris-HCl buffer, pH 7.5 and then resuspended in thesame buffer supplemented with 5 mM MgCl. 1 mM CaCl₂ and 0.1M NaCl. About5×10⁷ cells per assay are used.

Results of these assays using platelets and PMNCs are shown in Table V,demonstrating that the ReO complexes of the peptides of the inventionare potent antagonists to vasoactive intestinal receptor binding onthese peripheral blood cells.

                  TABLE V                                                         ______________________________________                                        Peptide      IC.sub.50  (nM): Platelets                                                                  IC.sub.50  (nM): PMNCs                             ______________________________________                                        vasoactive intenstinal                                                                     4.0           5.5                                                  peptide                                                                       P887 6 6.0                                                                    P915 4.5 7.5                                                                  P916 3.0 6.5                                                                  P917 0.8 2.7                                                                ______________________________________                                    

where vasoactive intestinal peptide is:

    HSDAVFTDNYTRLRKQMAVKKYLNSILN                               (SEQ ID NO:1).

The following tumor cell lines were assayed using the above-describedbinding competition assay: KU812 cells (human chronic myelogenousleukemia cell line): COLO320 cells (human colon adenocarcinoma cellline); HT29 cells (human colorecal adenocarcinoma cell line); PANC1cells (human pancreatic adenocarcinoma cell line); HMC1 cells (humanmast cells). Each cell line is assayed essentially as described abovefor human peripheral blood cells. Cells are grown in RPMI media orDulbecco's Modified Media (HMC1 cells) supplemented with 10% fetal calfserum, glutamine and antibiotics using standard cell culture techniques(see Animal Cell Culture: A Pratical Approach, Freshney, ed, IRL Press:Oxford, UK, 1992).

Results of these assays are shown in Table VI, demonstrating that theReO complexes of the peptides of the invention are potent inhibitors ofvasoactive intestinal binding to these human tumor cells.

                  TABLE VI                                                        ______________________________________                                                                     IC.sub.50                                             (nM):                                                                      Peptide KU812 COLO320 HT29 PANC1 HMC1                                       ______________________________________                                        vasoactive intestinal                                                                     0.8     1.5      1.2   4.2   7                                      peptide                                                                       P887 5.2 6.0 6.6 >10 8.5                                                      P915 N.D. 6.7 7.0 14 15                                                       P916 N.D. 4.0 8.5 12.5 7                                                      P917 1.0 2.0 2.2 3.7 7                                                      ______________________________________                                         where vasoactive intestinal peptide is HSDAVFTDNYTRLRKQMAVKKYLNSILN.amide

These results demonstrate that the vasoactive intestinal peptides andReO complexes thereof provided by the invention are capable ofspecifically binding to vasoactive intestinal receptors in standard invitro assays on a variety of normal and human tumor cell types. Theseresults indicate that the vasoactive intestinal peptides of theinvention have utility as scintigraphic imaging agents for imaging tumorsites in humans.

These assays are used to select effective radiotherapeutic agents asprovided by the invention, wherein the in vitro vasoactive intestinalreceptor-expressing cells and membranes are used to detect and quantifyvasoactive intestinal receptor binding of Re-complexed reagents of theinvention comprising vasoactive intestinal peptides covalently linked toa chelating moiety incorporated into the reagent during peptidesynthesis. Such Re-complexed reagents can then be evaluated incompetition binding assays as described herein using radioiodinatedvasoactive intestinal.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 6                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 28                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (28)                                                          <223> OTHER INFORMATION: AMIDATION                                             - - <400> SEQUENCE: 1                                                         - - His Ser Asp Ala Val Phe Thr Asp Asn Tyr Th - #r Arg Leu Arg Lys        Gln                                                                               1               5 - #                 10 - #                 15             - - Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Le - #u Asn                                   20     - #             25                                         - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (29)                                                          <223> OTHER INFORMATION: AMIDATION                                            <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (29)                                                          <223> OTHER INFORMATION: cys-29 is linked to a - #(BAT) chelator              <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:synthetic            peptide                                                                  - - <400> SEQUENCE: 2                                                         - - His Ser Asp Ala Val Phe Thr Asp Asn Tyr Th - #r Arg Leu Arg Lys Gln        1               5 - #                 10 - #                 15              - - Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Le - #u Asn Cys                               20     - #             25                                         - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 31                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:synthetic            peptide                                                                 <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (31)                                                          <223> OTHER INFORMATION: AMIDATION                                             - - <400> SEQUENCE: 3                                                         - - His Ser Asp Ala Val Phe Thr Asp Asn Tyr Th - #r Arg Leu Arg Lys Gln        1               5 - #                 10 - #                 15              - - Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Le - #u Asn Gly Gly Cys                       20     - #             25     - #             30                  - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 27                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (27)                                                          <223> OTHER INFORMATION: AMIDATION                                            <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:synthetic            peptide                                                                  - - <400> SEQUENCE: 4                                                         - - Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Ar - #g Leu Arg Lys Gln Met        1               5 - #                 10 - #                 15              - - Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu As - #n                                       20     - #             25                                         - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 20                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (20)                                                          <223> OTHER INFORMATION: AMIDATION                                            <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:synthetic            peptide                                                                  - - <400> SEQUENCE: 5                                                         - - Asn Tyr Thr Arg Leu Arg Lys Gln Met Ala Va - #l Lys Lys Tyr Leu Asn        1               5 - #                 10 - #                 15              - - Ser Ile Leu Asn                                                                       20                                                                - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 14                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (14)                                                          <223> OTHER INFORMATION: AMIDATION                                            <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence:synthetic            peptide                                                                  - - <400> SEQUENCE: 6                                                         - - Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Se - #r Ile Leu Asn                1               5 - #                 10                                   __________________________________________________________________________

We claim:
 1. A complex comprising:a) a radionuclide selected from thegroup consisting of technetium-99m, rhenium-186, and rhenium-188; and b)a synthetic, receptor-binding vasoactive intestinal peptide covalentlylinked to a technetium or rhenium chelating moiety;wherein the complexhas a vasoactive intestinal peptide receptor binding affinity not lessthan about one-tenth the affinity of radioiodinated native vasoactiveintestinal peptide for said receptor.
 2. The complex of claim 1, formedby reacting the synthetic peptide with technetium-99m, rhenium-186, orrhenium-188 in the presence of a reducing agent.
 3. The complex of claim2, wherein the reducing agent is a stannous ion.
 4. The complex of claim1, formed by ligand exchange between the synthetic peptide and aprereduced technetium-99m complex.
 5. The complex of claim 1, formed byligand exchange between the synthetic peptide and a prereducedrhenium-186 complex.
 6. The complex of claim 1, formed by ligandexchange between the synthetic peptide and a prereduced rhenium-188complex.
 7. A composition comprisinga) a synthetic, receptor-bindingvasoactive intestinal peptide covalently linked to a technetium orrhenium chelating moiety;wherein the synthetic peptide when radiolabeledhas a vasoactive intestinal peptide receptor binding affinity not lessthan about one-tenth the affinity of radioiodinated native vasoactiveintestinal peptide for said receptor; and b) a stannous ion.
 8. A methodof labeling a synthetic, receptor-binding vasoactive intestinal peptidecovalently linked to a technetium or rhenium chelating moiety comprisingthe step of reacting the synthetic peptide with technetium-99m,rhenium-186, or rhenium-188 in the presence of a reducing agent, whereinthe radiolabeled synthetic peptide has a vasoactive intestinal peptidereceptor binding affinity not less than about one-tenth the affinity ofradioiodinated native vasoactive intestinal peptide for said receptor.9. The method of claim 8, wherein the reducing agent is a stannous ion.10. A method of labeling a synthetic, receptor-binding vasoactiveintestinal peptide covalently linked to a technetium or rheniumchelating moiety by ligand exchange, comprising the step of reacting thesynthetic peptide with technetium-99m, rhenium-186, or rhenium-188 witha prereduced technetium-99m, rhenium-186, or rhenium-188 complex.
 11. Acomposition comprising a peptide having a formula selected from thegroup consisting of:

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(β-Dap)KCK.amide).amide;

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCK.amide).amide;

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(δ-Orn)GCK.amide).amide; and

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCE.amide).amide.


12. A kit comprising a sealed vial containing:a) a predetermined amountof a peptide having a formula selected from the group consisting of:

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(β-Dap)KCK.amide).amide;

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCK.amide).amide;

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(δ-Orn)GCK.amide).amide; and

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCE.amide).amide;

and b) a sufficient amount of a reducing agent to label the peptide withtechnetium-99m, rhenium-186, or rhenium-188.
 13. A method of imaging atumor within a mammalian body comprising the steps of:a) administeringto the body a technetium-99m-labeled peptide having a formula selectedfrom the group consisting of:

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(β-Dap)KCK.amide).amide;

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCK.amide).amide;

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(δ-Orn)GCK.amide).amide; and

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCE.amide).amide;

and b) detecting technetium-99m accumulated at the tumor.
 14. A methodof treating a tumor within a mammalian body comprising the step of:a)administering to the body a cytotoxic amount of a rhenium-186 orrhenium-188-labeled peptide having a formula selected from the groupconsisting of:

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(β-Dap)KCK.amide).amide;

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCK.amide).amide;

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.(δ-Orn)GCK.amide).amide; and

    HSDAVFTDNYTRLRKQMAVKKYLNSILNC(CH.sub.2 CO.GGCE.amide).amide.